Systems and methods for manufacturing a silk fibroin solution and powders containing silk fibroin

ABSTRACT

The disclosure relates to systems and methods for improving the manufacturing of silk solutions and powders containing silk fibroin obtained from silkworm cocoons. The solutions and powders can be used to improve the post-harvest preservation of perishables and to improve the performance of packaging, including biodegradable packaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.17/650,570, filed on Feb. 10, 2022, which claims priority to and thebenefit of U.S. Provisional Pat. Application No. 63/191,441, filed May21, 2021; U.S. Provisional Application No. 63/212,283, filed Jun. 18,2021; and U.S. Provisional Application No. 63/231,399, filed Aug. 10,2021, which applications are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The disclosure relates to systems and methods for improving themanufacturing of silk solutions containing silk fibroin from silk inputsand the manufacturing of silk fibroin powders derived therefrom.

BACKGROUND

One third of the food produced in the world is wasted each year and over45% of all fruits and vegetables are lost to spoilage. Food waste hasmassive economic, social, and environmental implications. According tothe Natural Resources Defense Council (NRDC), a prominent non-profitinternational environmental advocacy group, the United States loses 40%of its food supply resulting in an estimated economic loss of $165billion per year. Embodiments of the present disclosure directly addressthe broader societal need for reducing food waste and increasing foodavailability by extending the shelf-life of perishables (e.g., cooked oruncooked meats, proteins, carbohydrates, produce, nuts, grains, seeds,dairy, beverages, processed foods (e.g., chocolates, candies, chips,snacks, energy bars), gums, tablets, capsules, plants, roots, fungi,spores, breads, dried fruits, dried vegetables, dehydrated foods,medical foods, flowers, plants, and the like). Embodiments of thepresent disclosure represent significant commercial value by increasingrevenue through improved distribution, reducing waste, and decreasingcosts associated with cold storage and transport.

SUMMARY OF THE INVENTION

The disclosure relates to systems and methods for improving themanufacturing of silk solutions and powder containing silk fibroinobtained from silk inputs, which can be used to improve the post-harvestpreservation of perishables and to improve the performance of packaging,including biodegradable packaging.

In one embodiment, the disclosure provides a manufacturing process forsilk fibroin, where a silk source or silk input, such as silk cocoons(the silk cocoons can be whole, including the silkworm pupae, or beprocessed to remove the pupae and/or be cut in a specific manner), silksheets, silk floss, or silk pellets, cut cocoons, shredded cocoons, silkyarns and threads, silk textiles, silk powder, silk grinds, silkwadding, silk protein, degummed silk, silk mats, silk webbing, silkfibers, or the like, is processed into a solution or a powder thatincludes silk fibroin. For example, from a Bombyx mori silkworm is anexample of a silk source that may be used in this process. Thisdisclosure also applies to silk sources from silkworms other than theBombyx mori (e.g., Bombyx mandarina, Bombyx sinesis, Anaphe moloneyi,Anaphe panda, Anaphe reticulate, Anaphe ambrizia, Anaphe carteri, Anaphevenata, Anapha infracta, Antheraea assamensis, Antheraea assama,Antheraea mylitta, Antheraea pernyi, Antheraea yamamai, Antheraeapolyphemus, Antheraea oculea, Anisota senatoria, Apis mellifera, Araneusdiadematus, Araneus cavaticus, Automeris io, Atticus atlas, Copaxamultifenestrata, Coscinocera hercules, Callosamia promethea, Eupackardiacalleta, Eurprosthenops australis, Gonometa postica, Gonometarufobrunnea, Hyalophora cecropia, Hyalophora euryalus, Hyalophoragloveri, Miranda auretia, Nephila madagascarensis, Nephila clavipes,Pachypasa otus, Pachypasa atus, Philosamia ricini, Pinna squamosa,Rothschildia hesperis, Rothschildia lebeau, Samia cynthia, and Samiaricini, and Tetragnatha versicolor.), as well as spiders, or otherinsects. This disclosure also applies to silk sources generatedsynthetically, by genetic recombination, transgenically, and otherengineered silk (e.g., silks from bacteria, yeast, mammalian cells,transgenic animals, or transgenic plants). Silk proteins have a uniqueamino acid sequence repeatable via synthetic forms. This disclosurerelates to such forms. For the avoidance of doubt, silk cocoons asdescribed herein may be substituted for any of the above forms of silk,or similar forms of silk, be that natural or artificial. For example, ifthe disclosure states silk, silk inputs, silk cocoons, or silkwormcocoons are used, that means that any of the silk sources discussed inthis paragraph (e.g., cocoons, floss, sheets, pellets, cut cocoons,shredded cocoons, silk yarns and threads, silk textiles, silk powder,silk grinds, silk wadding, silk protein, degummed silk, silk mats, silkwebbing, silk fibers, generated silk sources (e.g., generatedsynthetically, by genetic recombination, transgenically, and otherengineered silk), etc.) or a combination thereof may be used. In oneembodiment, the silk cocoons are subjected to a degumming step, adissolution step, a purification step, a microfiltration step, and apowderization step, which results in a powder of the silk solutioncontaining silk fibroin. In some embodiments, the silk fibroin may beisolated from the silk cocoons through the Ajisawa method or throughother methods using water and salts, including chaotropic and/orkosmotropic agents. In some embodiments, silk fibroin may be preparedaccording to the method described in Marelli, B., Brenckle, M., Kaplan,D. et al. Silk Fibroin as Edible Coating for Perishable FoodPreservation. Sci Rep 6, 25263 (2016),https://doi.org/10.1038/srep25263, incorporated herein by reference inits entirety. The microfiltration step discussed herein would work withany acceptable method of isolating silk fibroin from silk cocoons,including instances where the silk fibroin is processed into a silksolution or as a powder. In some embodiments, the silk fibroin may be asdescribed in U.S. Pat. Publication No. 2020-0178576 A1, incorporatedherein by reference in its entirety.

In some embodiments the silk fibroin present in an aqueous solution orpowder may have a weight concentration (w/w) range from about 0.1% (w/w)to about 1% (w/w), 0.1% (w/w) to about 10% (w/w), 0.1% (w/w) to about30% (w/w), 0.1% (w/w) to about 50% (w/w), from about 1% (w/w) to about5% (w/w), from about 1% (w/w) to about 10% (w/w), from about 1% (w/w) toabout 15% (w/w), from about 5% (w/w) to about 10% (w/w), from 5% (w/w)to about 15% (w/w), from 5% (w/w) to about 20% (w/w), from 10% (w/w) toabout 30% (w/w), from 10% (w/w) to about 100% (w/w), from 50% (w/w) toabout 75% (w/w), from 10% (w/w) to about 100% (w/w), from about 20%(w/w) to about 95% (w/w), from about 30% (w/w) to about 90% (w/w), 30%(w/w) to about 100% (w/w), from about 40% (w/w) to about 85% (w/w), fromabout 50% (w/w) to about 80% (w/w), from about 60% (w/w) to about 99%(w/w), from about 70% (w/w) to about 99% (w/w), from about 80% (w/w) toabout 99% (w/w), from about 80% (w/w) to about 100% (w/w), from about90% (w/w) to about 99% (w/w), from about 95% (w/w) to about 99% (w/w),from about 90% (w/w) to about 100% (w/w), or from about 80% (w/w) toabout 90% (w/w). In an embodiment, the percent silk fibroin (w/w)present in an aqueous solution or powder is less than 99%. In anembodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 95%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 60%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 30%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 25%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 20%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 19%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 18%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 17%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 16%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 15%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 14%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 13%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 12%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 11%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 10%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 9%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 8%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 7%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 6%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 5%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 4%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 3%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 2%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than 1%.In an embodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 0.9%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than0.8%. In an embodiment, the percent silk fibroin (w/w) present in anaqueous solution or powder is less than 0.7%. In an embodiment, thepercent silk fibroin (w/w) present in an aqueous solution or powder isless than 0.6%. In an embodiment, the percent silk fibroin (w/w) presentin an aqueous solution or powder is less than 0.5%. In an embodiment,the percent silk fibroin (w/w) present in an aqueous solution or powderis less than 0.4%. In an embodiment, the percent silk fibroin (w/w)present in an aqueous solution or powder is less than 0.3%. In anembodiment, the percent silk fibroin (w/w) present in an aqueoussolution or powder is less than 0.2%. In an embodiment, the percent silkfibroin (w/w) present in an aqueous solution or powder is less than0.1%. Higher or lower silk fibroin content may also be possible to suita particular application, for example, method of application, type ofproduct to be coated, etc.

In some embodiments, the silk fibroin comprises silk fibroin monomers,polymers, and/or fragments. As used herein, the term silk fibroinfragments also include assemblies of silk fibroin fragments. In someembodiments, a silk film and/or coating can be formed from the silkfibroin and the silk film and/or coating comprises a specific percentage(weight/volume) of silk fibroin fragments. In some embodiments, aspecific percentage of the silk fibroin fragments have a specificmolecular weight (MW). In this context, molecular weight (MW) refers tothe molecular weight of individual silk fibroin fragments in a silk filmand/or coating, and is not to be confused with weight average molecularweight (M_(w)). To measure the various characteristics of the silk, onecould use any industry appropriate method or device. In one example, gelpermeation chromatography (GPC) could be used to acquire the molecularweight (MW) of silk fibroin fragments and the weight average molecularweight (M_(w)) of the silk.

As an illustrative example, FIGS. 12 and 13 illustrate two differentexemplary graphs of the molecular weights of silk fibroin fragmentspresent in a silk film and/or coating. The X axis represents molecularweight (MW), and the Y axis represents intensity (e.g., the number ofsilk fibroin fragments with the same molecular weight). The blue barillustrates a molecular weight (MW) range (e.g., 50 kDa to 100 kDa) thatincludes a certain percentage (e.g., 10%) of the fibroin fragments inthe silk film and/or coating, which is measured when the silk fibroinfragments are still in solution. The Figures also include peaks (P), forexample FIG. 12 has one peak and FIG. 13 has two peaks. As a furtherexample, a graph of the molecular weights (MW) of a silk film and/orcoating could include more than two peaks. For the purposes of thisdisclosure, the number of peaks is not limiting and does not impact thepercentages of silk fibroin fragments with a specific molecular weight(MW) as discussed herein. Molecular weights may also be measured viaother means, such as sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) or other similar techniques.

In some aspects, none of the silk fibroin fragments have a molecularweight (MW) under 100 kilodaltons (kDa), less than 1% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 1% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 5% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 10% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 15% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 20% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 25% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 30% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 35% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 40% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 45% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 50% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 55% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 60% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 65% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 70% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 75% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 80% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa, more than about 85% of the silkfibroin fragments have a molecular weight (MW) under 100 kDa, more thanabout 90% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, more than about 95% of the silk fibroin fragments have amolecular weight (MW) under 100 kDa.

In some aspects, none of the silk fibroin fragments have a molecularweight (MW) above 100 kDa, less than 1% of the silk fibroin fragmentshave a molecular weight (MW) above 100 kDa, more than about 1% of thesilk fibroin fragments have a molecular weight (MW) above 100 kDa, morethan about 5% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 10% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 15% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 20% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 25% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 30% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 35% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 40% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 45% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 50% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 55% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 60% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 65% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 70% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 75% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 80% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, more than about 85% of the silk fibroin fragments have amolecular weight (MW) above 100 kDa, more than about 90% of the silkfibroin fragments have a molecular weight (MW) above 100 kDa, more thanabout 95% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa.

In some aspects, none of the silk fibroin fragments have a molecularweight (MW) above 200 kDa, less than 1% of the silk fibroin fragmentshave a molecular weight (MW) above 200 kDa, more than about 1% of thesilk fibroin fragments have a molecular weight (MW) above 200 kDa, morethan about 5% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 10% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 15% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 20% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 25% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 30% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 35% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 40% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 45% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 50% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 55% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 60% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 65% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 70% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 75% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 80% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, more than about 85% of the silk fibroin fragments have amolecular weight (MW) above 200 kDa, more than about 90% of the silkfibroin fragments have a molecular weight (MW) above 200 kDa, more thanabout 95% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa.

In some aspects, none of the silk fibroin fragments have a molecularweight (MW) above 300 kDa, less than 1% of the silk fibroin fragmentshave a molecular weight (MW) above 300 kDa, more than about 1% of thesilk fibroin fragments have a molecular weight (MW) above 300 kDa, morethan about 5% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 10% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 15% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 20% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 25% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 30% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 35% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 40% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 45% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 50% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 55% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 60% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 65% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 70% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 75% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 80% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, more than about 85% of the silk fibroin fragments have amolecular weight (MW) above 300 kDa, more than about 90% of the silkfibroin fragments have a molecular weight (MW) above 300 kDa, more thanabout 95% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa.

In some aspects, none of the silk fibroin fragments have a molecularweight (MW) above 400 kDa, less than 1% of the silk fibroin fragmentshave a molecular weight (MW) above 400 kDa, more than about 1% of thesilk fibroin fragments have a molecular weight (MW) above 400 kDa, morethan about 5% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 10% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 15% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 20% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 25% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 30% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 35% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 40% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 45% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 50% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 55% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 60% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 65% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 70% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 75% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 80% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, more than about 85% of the silk fibroin fragments have amolecular weight (MW) above 400 kDa, more than about 90% of the silkfibroin fragments have a molecular weight (MW) above 400 kDa, more thanabout 95% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa.

In some aspects, between about 1% and about 10% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 1%and about 15% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 1% and about 30% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 10%and about 30% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 10% and about 50% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 10%and about 75% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 10% and about 95% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 15%and about 30% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 15% and about 40% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 20%and about 30% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 20% and about 35% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 30%and about 50% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 50% and about 90% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 50%and about 75% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 60% and about 75% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa, between about 75%and about 95% of the silk fibroin fragments have a molecular weight (MW)under 100 kDa, between about 80% and about 95% of the silk fibroinfragments have a molecular weight (MW) under 100 kDa.

In some aspects, between about 1% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 30%and about 90% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 40% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 50%and about 90% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 60% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 50%and about 85% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 60% and about 85% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 55%and about 80% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 65% and about 85% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 60%and about 80% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 70% and about 80% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 60%and about 99% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 70% and about 99% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa, between about 80%and about 99% of the silk fibroin fragments have a molecular weight (MW)above 100 kDa, between about 90% and about 99% of the silk fibroinfragments have a molecular weight (MW) above 100 kDa.

In some aspects, between about 0.1% and about 40% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 0.1%and about 30% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 0.1% and about 20% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 0.1%and about 10% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 0.5% and about 40% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 0.5%and about 30% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 0.5% and about 20% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 0.5%and about 10% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 1% and about 30% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 1%and about 20% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 1% and about 10% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 20%and about 80% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 40% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 50%and about 90% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa, between about 60% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 200 kDa, between about 60%and about 80% of the silk fibroin fragments have a molecular weight (MW)above 200 kDa.

In some aspects, between about 0.1% and about 3% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 0.1%and about 5% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 0.1% and about 10% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 1%and about 30% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 1% and about 10% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 1%and about 20% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 5% and about 20% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 10%and about 20% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 10% and about 30% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 10%and about 50% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 10% and about 75% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 10%and about 95% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 15% and about 30% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 20%and about 50% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 30% and about 50% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 50%and about 90% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 50% and about 75% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 60%and about 75% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa, between about 75% and about 95% of the silk fibroinfragments have a molecular weight (MW) above 300 kDa, between about 80%and about 95% of the silk fibroin fragments have a molecular weight (MW)above 300 kDa.

In some aspects, between about 1% and about 5% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 1%and about 10% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 1% and about 20% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 1%and about 30% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 1% and about 60% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 5%and about 10% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 5% and about 15% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 5%and about 20% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 30% and about 60% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 35%and about 55% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 35% and about 75% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 35%and about 85% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 50% and about 85% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa, between about 55%and about 80% of the silk fibroin fragments have a molecular weight (MW)above 400 kDa, between about 70% and about 90% of the silk fibroinfragments have a molecular weight (MW) above 400 kDa.

In one aspect, the disclosure relates to a silk manufacturing systemincluding multiple processing substations. Specifically, the systemincludes a first processing substation with a vessel configured toreceive silkworm cocoons and extract silk fibroin proteins therefrom toproduce a silk fibroin-based solution, a second processing substation influid communication with the first processing substation and configuredto receive and purify the silk fibroin-based solution from the firstprocessing substation, a third processing substation in fluidcommunication with the second processing substation and configured toreceive and sterilize the purified silk fibroin-based solution, and afourth processing substation in fluid communication with the thirdprocessing substation and configured to receive and powderize the silkfibroin-based solution. In various aspects, the systems disclosed hereinmay include any number and arrangement of processing substations asnecessary for a particular application.

In various embodiments of the foregoing aspect, the system furtherincludes a pump assembly disposed between the first and secondprocessing substations and configured to transfer the silk fibroin-basedsolution from the first processing substation to the second processingsubstation. The system may also include a reservoir disposed between thefirst and second processing substations and configured to at least oneof hold or condition the silk fibroin-based solution, such as, forexample, to adjust a temperature of the solution or adjust aconcentration of one or more components of the solution. Additionally,the system may further include a filtration system disposed between thefirst and second processing substations and configured to filter thesilk fibroin-based solution and a heat exchange system configured toadjust a temperature of the silk fibroin-based solution prior to orafter any one of the processing substations.

In further embodiments, the first processing substation is configured toextract the silk fibroin proteins via degumming, rinsing, and dissolvingprocesses within a single vessel. The second processing substation maybe configured to purify the silk fibroin-based solution and/orconcentrate the silk fibroin-based solution to have a higher percentageof silk fibroin via ultrafiltration and/or diafiltration, with orwithout the use of tangential flow filtration, or dialysis. The thirdprocessing substation may be configured to moderately clean or sterilizethe purified silk fibroin-based solution via one or more ofultrafiltration, microfiltration, pasteurization, or something similar.Generally, sterilization is not necessarily intended to include asolution completely free from bacteria or other living microorganisms,but it could be. Another substation may be centrifugation ormicrofiltration to reduce turbidity. Excess turbidity may be undesirablein a silk fibroin-based solution, as it may impact the tackiness of acoating made from the silk fibroin-based solution, hinder the barrierforming properties of the silk solution, and/or may cause a coatingformed from the silk fibroin-based solution to look cloudy or milky. Forthis reason, turbidity may be kept under about 1.000 optical density,including in solution concentrations of 2.5%, 5%, 7.5, 10%, 12.5%, 15%,17.5%, or 20% silk fibroin-in-water, wherein the optical density ismeasured at a wavelength of 660 nm (OD660). In some embodiments, theturbidity may be kept under a lower limit, such as about 0.900, 0.800,0.700, 0.600, 0.500, 0.400, 0.300, 0.200, 0.100, 0.050 (OD600), or inany increments within.

Additionally, the presence of excessive amounts of microbes maynegatively impact the performance of the silk solution and potentiallymake the silk fibroin-based solution unfit for human consumption ortarget application, including in pre-harvest applications, post-harvestapplications, animal-feed applications, or other such applications. Forthis reason, microbes should be killed and/or substantially removed fromthe silk fibroin-based solution, which may range from a small level ofreduction to essentially complete removal as, for example, may bedetermined within the limitations of detection and/or the type ofmicrobes (e.g., under 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 cfu/gfor mold, yeast, Enterobacteriaceae, Staphylococcus aureus, Escherichiacoli, and a “Negative/under 25 g” reading for Salmonella and Listeriamonocytogenes). In one example, this may mean keeping the amounts ofmicrobes under 10 CFU/ml under any acceptable testing mechanism, forexample total aerobic plate count on plate count agar (PCA) and/orpotato dextrose agar (PDA). In some cases, the third processingsubstation is configured to sterilize the purified silk fibroin-basedsolution to a food grade standard. In some cases, the third processingsubstation may be removed entirely from the system. In those cases, orother cases, the previous substations may produce a food grade standardproduct and/or sterilize the silk-fibroin based solution to the leveldiscussed herein. For example, the first substation may sterilize thesilk-fibroin based solution by treating the silk fibroin-based solutionat a certain temperature to remove microbes from the solution. In thisexample, the entire system could alternatively be closed, so that nofurther sterilization is necessary. In other cases, the third processingsubstation may be placed at different locations in the system. In somecases, the entire system may be a closed system such that microbes maynot be present in large enough numbers to necessitate the thirdprocessing substation. Furthermore, the fourth processing substation maybe configured to powderize the purified silk fibroin-based solution viaspray drying, freeze drying, or similar drying and powderization methodsknown in the industry.

In still other embodiments, the system may further include apre-treatment system configured to condition the silkworm cocoons priorto or at introduction to the first processing substation, such as, forexample, a shredder for shredding the silkworm cocoons, softeningequipment, soaking equipment, and/or material handling equipment. Insome embodiments, the silkworm cocoons are shredded to a reduced sizeand shape (e.g., 0.5-50 cm fragments or 0.5-50 cm strands of longer silkfloss, sheets, or wadding) and/or treated or pressed. In someembodiments, prior to introduction into the system, the cocoons or othersilk inputs may be stripped of sericin, washed to remove organic andinorganic compounds, stripped of other proteins, or combined with morethan one silk input to increase the amount of fibroin per unit mass ofsilk input. This may or may not include shredding or cutting or apreliminary degum step. In addition, the pretreatment equipment mayinclude systems for cleaning the cocoons, including separating debrisfrom the cocoons, testing the cocoons (e.g., chemical analysis), and/orperforming other quality control processes, including cocoon compositionassessments.

The system may also include a post-treatment system configured toreceive the silk fibroin powder from the fourth processing substation.The post-treatment equipment may include equipment conditioning the silkfibroin powder by the addition of one or more additives or silk powderfrom a different batch with different chemical or polymercharacteristics (i.e., molecular weight profiles, turbidities, or thelike) (e.g., lower molecular weight silk fibroin may be added to highermolecular weight silk fibroin to allow for an increase in instantizationand solubility or to allow for different characteristics andproperties). The post-treatment equipment may also add a heat treatmentstep or an agglomeration step that may make the powder dryer, wetter,denser, cleaner, and/or more instantizable. The post-treatment equipmentmay also include equipment for testing the silk fibroin powder and/orpackaging the silk fibroin powder. The post-treatment step may be anaseptic method of packaging to allow for shelf-stable silk fibroinpowder.

The system may include a controller in communication with the variousprocessing substations (e.g., valve assemblies, sensors, switches,transmitters, drives, etc.) and configured to control one or more of theintroduction variables (e.g., volumes, flow rates, mixing rates,agitation speeds, timing/duration of a process, pre-processingoperations, component proportions, pH levels, temperatures, pressures,solution amounts, solids amounts, etc.) of the various components (e.g.,cocoons, solvents, compounds, etc.), controlling a degumming operation(e.g., soak times and temperatures, pressurization, agitation speeds andtiming thereof, volume control (i.e., draining and refilling vessel,recirculation)), controlling a rinse operation (e.g., determining stateof solution, draining and refilling of the vessel, addition of asolvent, frequency and duration of the various steps, pressurization, ordepressurization), controlling the silk fibroin dissolving operation(e.g., addition of the second compound and concentration thereof, time,temperature, pressure, agitation speeds and timing thereof, duration,etc.), controlling outputs from the substations (e.g., flow rates,temperatures, etc.).

In various embodiments of any of the aspects disclosed herein, the firstprocessing substation includes a reactor vessel having a first inletport configured to receive the silkworm cocoons and one or moreingredients (e.g., soda ash, a chaotropic agent, a catalyst, additive,or similar), a second inlet port configured to receive a solvent (e.g.,water, ethanol, citric acid, etc.) and at least one outlet configured tooutput the silk fibroin-based solution. The reactor vessel is configuredto process the silkworm cocoons by at least one of degumming, rinsing,and dissolving the silk fibroin protein from the cocoons. The firstprocessing substation may also include a water or oil jacket disposedabout the reactor vessel that is configured to provide heat exchange(e.g., heating or cooling as necessary) with the vessel and itscontents. The first processing substation may further include equipmentconfigured to agitate the contents of the reactor vessel, such as, forexample, a mixer, a vibration plate, a magnetic stirrer, sonicator,liquid pumps, air pumps, aqueous streams, etc. The agitation may occurthrough pressure streams external or internal, where the pressurestreams are liquid and/or gasses. In various embodiments, the agitationequipment may be disposed proximate a bottom or top surface of thereactor vessel. In various embodiments, the agitation equipment may bedisposed in various portions of the reactor vessel (i.e., pumps at thebottom, center, and top; agitator at the bottom and pump at the top;etc.). In some embodiments, the agitation equipment is a mixer having aunitary shaft and impeller. The impeller may be configured for axialflow, radial flow, and/or tangential flow, and may be run in reverse.Additionally, the impeller may be coated with a substance to resistattachment of silk fibers and/or have a surface finish of the blades(e.g., a surface roughness below some threshold value). The mixer mayhave interchangeable impellers, where the impellers may be configured tosuit particular processes and have one or more of flat blades, curvedblades, pitched blades, finger blades, anchor blades, gate blades,ribbon blades, etc. having different shapes, pitch, etc. The impellermay also be configured to be raised and lowered into the vessel orwithin the vessel contents during or between different processing steps.

In further embodiments, the reactor vessel includes a second outlet forremoving at least a portion of the solvent and any residue therein(e.g., dissolved sericin), which can be sent to waste, recirculated, orrecycled. The reactor vessel may have a glass lining and be sized tohave an aspect ratio of 0.5-5.0, or more preferably 0.8-2, and morepreferably 1.0-1.5 of height to diameter as defined by a work volume.The aspect ratio may be selected to suit a particular application, forexample, temperature control, processing rates, desired volumes, workspace, etc. The volume of the vessel will vary to suit a particularapplication (e.g., finished yields) and may range from about 0.25 litersto about 80.000 liters depending on the batch size required, preferably0.5 liters to 5.000 liters. Additionally, the reactor vessel may haveshapes other than cylindrical, such that the aspect ratio will be thevessel height to cross-sectional area (e.g., rectangular, ovoid, etc.cross-sectional shapes) thereof. The vessel contents may include aplurality of silkworm cocoons (with or without pre-treatment), a solvent(e.g., water), and a compound. The packing density of the silkwormcocoons will vary to suit a particular application (e.g., finished silkfibroin-based solution) and/or different silk inputs (e.g., cocoons,floss, etc.) and may range from: about 1%-100%, about 1%-70%, about1%-50%, about 1%-30%, about 1%-20%, about 2%-20%, about 2%-15%, lessthan about 100%, less than about 90%, less than about 80%, less thanabout 70%, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, greater thanabout 1%, greater than about 5%, greater than about 10%, greater thanabout 15%, greater than about 20%, greater than about 30%, greater thanabout 40%, greater than about 50%, greater than about 60%, greater thanabout 70%, greater than about 80%, greater than about 90%. The water oroil jacket is configured to heat the contents to a temperature of about50° C. to about 150° C., preferably about 75° C. to about 125° C., orother temperature to suit a particular application. In addition, therinse step may include performing 1 to 30 rinse cycles, more preferablyabout 1 to about 10 rinse cycles, more preferably about 3 to about 10rinse cycles, and more preferably about 4 to about 6 rinse cycles, oressentially any number of rinse cycles to suit a particular application.Generally, the process times, temperatures, pH, and other solutioncharacteristics may vary to suit a particular application, such as thetype of silk, or a particular output specification.

The reactor vessel may also include a handling structure or equipmentconfigured to control the movement and/or the position of the silkwormcocoons within the vessel (e.g., prevent floating of the cocoons). Theequipment may include, for example, a screen or netting disposedproximate a lower portion of the vessel and configured to separate thesilkworm cocoons from the agitation equipment and/or prevent thesilkworm cocoons from floating to the top of the vessel, a chute orfunnel structure in communication with the first inlet and configured todirect the silkworm cocoons to a particular location within the vesselduring introduction thereof, a recirculation system configured to draw aportion of the solution from a lower portion of the vessel andreintroduce the solution to an upper portion of the vessel and/orintroduce fresh water to push the silkworm cocoons down into thesolution, a vertically moveable sieve (e.g., a perforated plunger)disposed within the vessel and configured to “push” any solids withinthe solution towards a lower portion of the vessel, and one or morebaffles disposed within the vessel and extending from an inner wallthereof, where the baffles direct the movement of the solution andcontents therein. In some embodiments, the movement of the silkwormcocoons may be controlled by adjusting the processing temperaturesduring various stages of the process. For example, during a degummingoperation, the contents may be heated to a temperature slightly lowerthan their boiling point to reduce the formation of air bubbles. Othermanners of controlling movement of the cocoons are described in greaterdetail below. See, for example, FIGS. 9-11 .

The first processing substation, and system generally, may include oneor more valve assemblies (with manual or automatic actuators) that areconfigured to control the introduction and removal from the firstprocessing substation and/or the reactor vessel of any component, suchas, for example, silkworm cocoons, compounds, solvents, waste solutions,residues, and final silk fibroin-based solutions. The first processingsubstation, and system generally, may include at least one sensorconfigured to sense one or more of solution temperatures,concentrations, flow rates, pH, fluid levels, turbidity, particle size,molecular weight, pressurization, etc., which may be used to control(with or without human intervention) the operation of the variousprocesses.

In further embodiments of any of the aspects disclosed herein, thesecond processing substation includes a filtration module housing atleast one membrane. The filtration module has an inlet configured toreceive the silk fibroin-based solution including a second compound(e.g., a chaotropic agent, such as: calcium bromide; magnesium chloride;lithium acetate; lithium perchlorate; guanidinium chloride; ethanol;methanol; urea; thiourea; sodium dodecyl sulfate; lithium thiocyanate(LiSCN); sodium thiocyanate (NaSCN); calcium thiocyanate (Ca(SCN)₂);magnesium thiocyanate (Mg(SCN)₂); anhydrous or dihydrate calciumchloride (CaCl2); lithium chloride (LiCl); lithium bromide (LiBr); zincchloride (ZnCl₂); copper nitrate (Cu(NO₂)₂); copper ethylene diamine(Cu(NH₂CH₂CH₂NH₂)₂ (OH)₂); Cu(NH₃)₄(OH)₂; Ajisawa’s reagent(CaCl₂/ethanol/water); isopropanol; 1-butanol; 2-butanol; ethyl acetate;calcium nitrate; magnesium nitrate; calcium perchlorate; calciumchlorate; calcium acetate; dicalcium phosphate/calcium hydrogenphosphate; calcium sulfate; calcium fluoride; ammonium fluoride;ammonium sulfate; ammonium phosphate; diammonium phosphate (diammoniumhydrogen phosphate); ammonium dihydrogen phosphate; ammonium acetate;ammonium chloride; ammonium bromide; ammonium nitrate; ammoniumchlorate; ammonium iodide; ammonium perchlorate; ammonium thiocyanate;potassium fluoride; potassium sulfate; monopotassium phosphate;dipotassium phosphate (potassium hydrogen phosphate); tripotassiumphosphate; potassium acetate; potassium chloride; potassium bromide;potassium nitrate; potassium chlorate; potassium iodide; potassiumperchlorate; potassium thiocyanate; sodium fluoride; sodium sulfate;sodium monophosphates (e.g., monosodium phosphate, disodium phosphate,trisodium phosphate); sodium di- and polyphosphates (e.g., monosodiumdiphosphate, disodium diphosphate, trisodium diphosphate, tetrasodiumdiphosphate, sodium triphosphate); sodium acetate; sodium chloride;sodium bromide; sodium nitrate; sodium chlorate; sodium iodide; sodiumperchlorate; lithium fluoride; lithium sulfate; lithium phosphate;lithium chloride; lithium bromide; lithium nitrate; lithium chlorate;lithium iodide; magnesium fluoride; magnesium sulfate; monomagnesiumphosphate; mimagnesium phosphate; trimagnesium phosphate; magnesiumacetate; magnesium bromide; magnesium chlorate; magnesium iodide;magnesium perchlorate; magnesium thiocyanate; monocalcium phosphate;tricalcium phosphate; octacalcium phosphate; dicalcium diphosphate;calcium triphosphate; calcium iodide; guanidinium nitrate; guanidiniumiodide; guanidinium thiocyanate, or a combination thereof), an outletconfigured to output a purified silk fibroin-based solution with areduced concentration of any chaotropic agent (i.e., the retentate), anda waste port configured to output a portion of the second compound(i.e., the permeate). The filtration module is configured to remove thesecond compound from the silk fibroin-based solution via diafiltrationor dialysis. In some cases, the flow through the module is tangential toa surface of the membrane. The silk fibroin-based solution may alsoexperience some level of concentration that may be tuned to optimize alater process (e.g., sterilization or powderization). The silkfibroin-based solution may be circulated through the filtration modulefor a duration defined by about 1 diavolumes to about at least 12diavolumes, preferably about 3 diavolumes to about 10 diavolumes, andmore preferably about 5 diavolumes to about 9 diavolumes. In some cases,the concentrations levels of the chaotropic agent in the retentateand/or the pressure drop across the filtration module may also bemonitored to determine a state of the process. Generally, it isdesirable to obtain a level of remaining chaotropic agent that isvirtually undetectable to a user (e.g., tasteless); however, this levelwill vary for different agents and/or product applications and mayinclude less than 1,000 parts per million (ppm), less than 900 ppm, lessthan 650 ppm, less than 400 ppm, less than 300 ppm, less than 250 ppm,and even as low as under 150 ppm. In some cases, other tests areconducted to ensure that no contaminants or unwanted materials arepresent in the silk fibroin-based solution.

Additionally, the filtration module may include one or more spiral woundmembranes; however, other membrane structures, such as plate and frame,hollow fiber, etc., may be used to suit a particular application (e.g.,flow rates, pressures, etc.). The filtration module may include multiplestages and may include about one to about ten membranes, about one toabout eight membranes, about three to about eight membranes, about threeto about five membranes. Where multiple filter stages or filtrationmodules are used, the silk fibroin-based solution may pass therethroughin series, parallel, or both to suit a particular application. Thenumber, size, and configuration of the membranes will be selected basedon the various system parameters (e.g., flow rates). The structures andchemistries of the membrane active layers will also vary to suit aparticular application and may be structured with a molecular weightcut-off of about 1 kDa to about 300 kDa, about 1 kDa to about 100 kDa,about 1 kDa to about 50 kDa. Additionally, the membranes may bemanufactured from polyether sulfone (PES), polyvinylidene fluoride(PVDF), polyacrylonitrile (PAN), polypropylene (PP), polyethyleneterephthalate (PET), or combinations thereof.

The second processing substation may also include a heat exchangesystem, including any valves, pumps, controls, etc. as needed to controla temperature of the silk fibroin-based solution during processing. Forexample, lowering the temperature of the silk fibroin-based solutionprior to introduction to the filtration module may enhance the removalof the second compound. The second processing substation may alsoinclude one or more valve assemblies configured to direct the silkfibroin-based solution output with a reduced second compound to at leastone of the inlet (recirculation) or to the third processing substationand one or more sensors (e.g., differential pressure, temperatures, flowrates, a salinometer, conductivity, etc.) in communication with thecontroller. In some embodiments, the filtration module may include arecycling circuit for recovering the removed second compound, such as byevaporation.

In additional embodiments of any of the aspects disclosed herein, thethird processing substation includes a microfiltration module having aninlet configured to receive the purified silk fibroin-based solutionfrom the second processing substation and an outlet configured to outputa sterile silk fibroin-based solution. The microfiltration module isconfigured to reduce turbidity and/or remove microbes from the purifiedsilk fibroin-based solution. In some embodiments, the inlet isconfigured to receive the silk fibroin-based solution from the firstprocessing substation and the outlet is configured to output a sterilesilk fibroin-based solution to the second processing substation.Additionally, the microfiltration module may include one or more filterstages, with or without pumps, valves, and holding tanks as necessary.In some embodiments, the first filter stage may be disposed upstream ofthe second processing substation and the second filter stage may bedisposed downstream of the second processing substation. In embodimentsincluding one or more pumps, the pumps are configured to transfer thesilk fibroin-based solution between filter stages and/or processingsubstations and/or to another process as necessary after completing themicrofiltration process. In addition, one or more holding tanks may beincluded to store the solution or provide additional processing, such astemperature control or concentration adjustment, as may be necessary toaddress turbidity or sterility levels.

The filter stages may include one or more spiral wound membranes;however, other membrane structures, such as plate and frame, hollowfiber, bag filters, cartridges, etc., may be used to suit a particularapplication. In some embodiments, the microfiltration module may includetwo (2) stages, where the first stage is configured to remove largeaggregates, while the second stage is configured to remove smalleraggregates, and/or to sterilize and reduce the turbidity of thesolution. The membranes in the first stage may be configured for depthor surface filtration, with a pore size ranging from 0.65 - 15um. Themembranes in the second stage may be configured for depth or surfacefiltration, with a pore size ranging from about 0.05 - 0.65 µm. Themembranes may be made from PES, PP, or cellulose, with or without a foodgrade filtering aid. The filter stages may include about 1 to about 52membranes.

The silk fibroin-based solution may pass through the membranes inseries, parallel, or both to suit a particular application. Themembranes may have an average pore size of about 0.02 µm to about 15 µm.In some embodiments, the membranes in a first filter stage may have apore size in the range of about 0.7 µm to about 5 µm, preferably about0.9 µm and about 1.4 µm, while the membranes in a second filter stagemay have a pore size in the range of about 0.05 µm to about 0.8 µm,preferably about 0.2 µm to about 0.8 µm, where the silk fibroin-basedsolution passes through the first filter stage prior to passing throughthe second filter stage (e.g., to filter out larger aggregates in thefirst stage). In some cases, the silk fibroin-based solution containsminimal amounts of a chaotropic agent. Additionally, or alternatively,the third processing substation may include a heat exchange circuit tosterilize the solution via pasteurization.

In still further embodiments of any of the aspects disclosed herein, thefourth processing substation includes powderization equipment configuredto receive the sterilized silk fibroin-based solution from the thirdprocessing substation and output the silk fibroin protein in a powderform. In addition, the resulting powdered silk fibroin may have a wateractivity level below 1.0, 0.95, 0.9, 0.85, 0.8, 0.75. 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1. Preferably, the water activity level is under 0.9 toallow for a shelf-stable powder from a food and microbiologicalstandpoint. The powderization equipment may include a spray dryer havingan inlet configured to receive the sterilized silk fibroin-basedsolution from the third processing substation and an outlet configuredto output the silk fibroin protein in a powdered and easilyinstantizable form. In one embodiment, a spray dryer may be configuredto have a high pressure nozzle, where the spray is created by forcingfeed, in this case silk fibroin-based solution, through a nozzleorifice. Alternatively, a two-fluid nozzle spray dryer may be used,where the spray is created by the interaction between the feed andcompressed air. In a two-fluid nozzle configuration, the feed may beatomized via contact with compressed air with or without a subsequentnozzle heating step. Hot drying gases may also be used to accelerate theatomization engine when it meets the feed. The hot drying gases may beconfigured to travel at a low velocity. Other spray dryer configurationsmay be also used. As a non-limiting example, the spray dryer may be oneof the following types: high pressure nozzle, two-fluid nozzle,combustion nozzle, atomization.

Instantizable may encompass a range of characteristics, including butnot limited to a powder that is flowable and easily dispersible in aliquid to form a stable dispersion in the liquid without stirring orshaking the powder in the liquid, but that could alternatively becreated by stirring or shaking the powder in the liquid for only a shortperiod of time. In one embodiment, the moisture content of the powdershould be between about 1%-10%, more preferably between about 1.0%-7%.The fourth processing substation may also include a feed vessel forholding the sterilized silk fibroin-based solution prior to processing.The feed vessel may be configured treat the sterilized silkfibroin-based solution prior to processing to, for example, enhancepowderization or produce a more instantizable powder. The fourthprocessing substation may also include equipment disposed downstream ofthe powderization equipment for modifying the powdered silk fibroinprotein (e.g., inclusion of an additive to make it more instantizable,or equipment to assist with agglomeration) or packaging equipment. As anexample of agglomeration equipment, the fourth processing substation mayinclude an external fluid bed or a fluid bed integrated with thepowderization equipment. The agglomeration equipment may aid inagglomeration of the powdered silk fibroin protein, which may improvedispersibility, instantization, or wettability properties of thepowdered silk fibroin protein. Any suitable agglomeration equipment maybe utilized. In some embodiments, the powdered silk fibroin may bepassed through the agglomeration equipment after it is powderized. Inother embodiments, the agglomeration equipment may be integrated intothe spray dryer such that agglomeration occurs during the powderizationprocess. In some embodiments, the agglomeration equipment may increasethe size of the powdered silk fibroin protein particles by more thanabout 5%, more than about 10%, more than about 20%, more than about 30%,more than about 40%, more than about 50%, more than about 60%, more thanabout 70%, more than about 80%, more than about 90%, more than about100%, more than about 150%, more than about 200%, more than about 250%,more than about 300%, more than about 350%, more than about 400%, morethan about 500%, more than about 600%, more than about 700%, more thanabout 800%, more than about 900%, more than about 1000%.

In another aspect, the disclosure relates to a method of processingsilkworm cocoons to obtain food grade silk fibroin. The method includesthe steps of introducing a plurality of silkworm cocoons to a reactorvessel, introducing a solvent (e.g., water (e.g., softened water,filtered water, deionized water, tap water), ethanol, citric acid, orother suitable substances with an acidic pH) to the reactor vessel,introducing a first compound to the reactor vessel, introducing heat tothe contents of the reactor vessel to promote degumming of the silkwormcocoons, optionally pressurizing the reactor vessel and/or optionallyagitating the contents of the reactor vessel to control movement of thesilkworm cocoons within the reactor vessel, removing at least a portionof the solvent and any degumming residue if any, rinsing the degummedsilk fibroin, introducing a second compound to the reactor vessel (withor without additional solvent) to dissolve the remaining silk fibroinproteins in to the solution, filtering the contents of the reactorvessel to substantially remove the second compound (e.g., as necessaryto meet a specific level or range of purity) and produce a purified silkfibroin-based solution, directing the purified silk fibroin-basedsolution to a sterilization process to obtain a “food grade” qualitysilk fibroin-based solution, and powderizing the purified silkfibroin-based solution to obtain the silk fibroin in a powder form.Various parameters of the process will vary to suit a particularapplication, for example, the order of, quantities, and rates ofintroduction or removal of various components (e.g., silkworm cocoons,solvent, compounds, rinse solutions, etc.), operating temperatureranges, processing times (e.g., speed and timing of agitation step(s)),order of operation, etc. In various embodiments, the methods disclosedherein may incorporate any of the additional processes or steps thatcorrespond to the systems and substations disclosed herein.

Embodiment 1: A silk manufacturing system comprising (A) a firstprocessing substation comprising a vessel configured to receive silkinputs, extract silk fibroin proteins therefrom, and produce a silkfibroin-based solution, such that the silk fibroin-based solution issubstantially free of sericin, wherein the first processing substationis configured to extract the silk fibroin proteins via degumming,rinsing, and dissolving processes within a single vessel; (B) a secondprocessing substation in fluid communication with the first processingsubstation, the second processing substation configured to receive andpurify the silk fibroin-based solution from the first processingsubstation, wherein the purified silk fibroin-based solution comprisesless than about 650 parts per million (ppm) of one or more salts ornon-organic particulates; (C) wherein the silk fibroin-based solution issterilized to produce a sterilized silk fibroin-based solution prior toa third processing substation; and (D) a third processing substation influid communication with the second processing substation, the thirdprocessing substation is a spray dyer that is configured to receive andpowderize the sterilized silk fibroin-based solution.

Embodiment 2: A silk manufacturing system comprising (A) a firstprocessing substation comprising a vessel configured to receive silkinputs, extract silk fibroin proteins therefrom, and produce a silkfibroin-based solution, wherein the first processing substation isconfigured to extract the silk fibroin proteins via degumming, rinsing,and dissolving processes within a single vessel; (B) a second processingsubstation in fluid communication with the first processing substation,the second processing substation configured to receive and purify thesilk fibroin-based solution from the first processing substation; (C) athird processing substation in fluid communication with the secondprocessing substation, the third processing substation configured toreceive and sterilize the purified silk fibroin-based solution; and (D)a fourth processing substation in fluid communication with the thirdprocessing substation, the fourth processing substation configured toreceive and powderize the purified silk fibroin-based solution, whereinthe fourth processing substation is a spray dryer.

Embodiment 3: A silk manufacturing system comprising (A) a firstprocessing substation configured to receive silk inputs, extract silkfibroin proteins therefrom, and produce a silk fibroin-based solution,wherein the first processing substation is configured to extract thesilk fibroin proteins within a single vessel, the first processingsubstation comprising a reactor vessel comprising a first inlet portconfigured to receive the raw silk inputs and one or more compounds, asecond inlet port configured to receive a solvent, and at least oneoutlet configured to output the silk fibroin-based solution, wherein thereactor vessel is configured to process the silk inputs by degumming,rinsing, and dissolving the silk fibroin protein from the silk inputs, aliquid jacket disposed about the reactor vessel and configured toprovide heat exchange with the vessel and its contents, wherein theliquid jacket is configured to heat the contents to a temperature ofabout 50° C. to about 150° C., and an agitation mechanism configured toagitate the contents of the reactor vessel; (B) a second processingsubstation in fluid communication with the first processing substation,the second processing substation configured to receive and purify thesilk fibroin-based solution from the first processing substation,wherein the second processing substation is configured to purify thesilk fibroin-based solution via tangential flow filtration, the secondprocessing substation comprising a filtration module housing at leastone membrane, the module comprising an inlet configured to receive thesilk fibroin-based solution including a compound, an outlet configuredto output a purified silk fibroin-based solution with a reduced compoundamount, and a waste port configured to output a portion of the compound,wherein the filtration module is configured to remove the compound fromthe silk fibroin-based solution by circulating the silk fibroin-basedsolution through the filtration module until about 1 diavolume to aboutat least 12 diavolumes are reached; (C) wherein the silk fibroin-basedsolution is sterilized to produce a sterilized silk fibroin-basedsolution prior to a third processing substation; (D) the thirdprocessing substation in fluid communication with the second processingsubstation, the third processing substation configured to receive andpowderize the sterilized silk fibroin-based solution, wherein the thirdprocessing substation is configured to powderize the sterilized silkfibroin-based solution via a spray dryer, and wherein the third processsubstation includes a piece of agglomeration equipment; and (E) apost-treatment system configured to receive a silk fibroin powder fromthe third processing substation and to at least one of: condition thesilk fibroin powder, test the silk fibroin powder, or package the silkfibroin powder in a food-safe container.

Embodiment 4: A silk manufacturing system comprising a first processingsubstation comprising a vessel configured to receive silk inputs,extract silk fibroin proteins therefrom, and produce a silkfibroin-based solution, such that the silk fibroin-based solution issubstantially free of sericin, wherein the first processing substationis configured to extract the silk fibroin proteins via degumming,rinsing, and dissolving processes within a single vessel.

Embodiment 5: The silk manufacturing system of any one of Embodiments 1to 4, or any combination thereof, wherein the purified silkfibroin-based solution comprises less than about 400 ppm of the one ormore salts or non-organic particulates.

Embodiment 6: The silk manufacturing system of any one of Embodiments 1to 5, or any combination thereof, wherein the powderized silkfibroin-based solution comprises a water activity level of less than0.9.

Embodiment 7: The silk manufacturing system of any one of Embodiments 1to 6, or any combination thereof, wherein the silk inputs come from aBombyx mori silkworm.

Embodiment 8: The silk manufacturing system of any one of Embodiments 1to 7, or any combination thereof, further comprising a reservoirdisposed between the first and second processing substations andconfigured to at least one of hold or condition the silk fibroin-basedsolution and a pump assembly disposed between the first and secondprocessing substations and configured to transfer the silk fibroin-basedsolution between the first processing substation, the reservoir, and thesecond processing substation.

Embodiment 9: The silk manufacturing system of any one of Embodiments 1to 8, or any combination thereof, wherein the second processingsubstation includes at least one spiral wound filtration membrane.

Embodiment 10: The silk manufacturing system of any one of Embodiments 1to 9, or any combination thereof, further comprising a heat exchangesystem configured to adjust a temperature of the silk fibroin-basedsolution prior to or after any one of the processing substations.

Embodiment 11: The silk manufacturing system of any one of Embodiments 1to 10, or any combination thereof, wherein the second processingsubstation is configured to purify the silk fibroin-based solution viadiafiltration.

Embodiment 12: The silk manufacturing system of any one of Embodiments 1to 11, or any combination thereof, wherein the second processingsubstation is configured to purify the silk fibroin-based solution viatangential flow filtration.

Embodiment 13: The silk manufacturing system of any one of Embodiments 1to 12, or any combination thereof, wherein the third or fourthprocessing substation includes a piece of agglomeration equipment.

Embodiment 14: The silk manufacturing system of any one of Embodiments 1to 13, or any combination thereof, further comprising a post-treatmentsystem configured to receive a silk fibroin powder from the thirdprocessing substation and to at least one of: condition the silk fibroinpowder, test the silk fibroin powder, or package the silk fibroin powderin a food-safe container.

Embodiment 15: The silk manufacturing system of any one of Embodiments 1to 14 or any combination thereof, wherein the third processingsubstation is configured to sterilize the purified silk fibroin-basedsolution to a food grade standard via microfiltration.

Embodiment 16: The silk manufacturing system of any one of Embodiments 1to 15 or any combination thereof, wherein the third processingsubstation is configured to sterilize the purified silk fibroin-basedsolution to a food grade standard via pasteurization.

Embodiment 17: The silk manufacturing system of any one of Embodiments 1to 16 or any combination thereof, further comprising a fourth processingsubstation in fluid communication with the second processing substation,the fourth processing substation configured to receive and sterilize thepurified silk fibroin-based solution.

Embodiment 18: The silk manufacturing system of any one of Embodiments 1to 17 or any combination thereof, wherein the fourth processingsubstation comprises a microfiltration module configured to receive atleast one of the silk fibroin-based solution or the purified silkfibroin-based solution and to remove microbes and reduce turbidity fromthe at least one of the silk fibroin-based solution or the purified silkfibroin-based solution.

Embodiment 19: The silk manufacturing system of any one of Embodiments 1to 18 or any combination thereof, wherein the microfiltration moduleincludes two filter stages, the first filter stage having a pore sizebetween about 0.7 µm and about 5 µm and the second filter stage has apore size between about 0.05 µm and about 0.8 µm, and the silkfibroin-based solution passes through the first filter stage prior topassing through the second filter stage.

Embodiment 20: The silk manufacturing system of any one of Embodiments 1to 19 or any combination thereof, wherein the microfiltration modulefurther comprises one or more pumps configured to transfer the silkfibroin-based solution between filter stages, between processingsubstations, to another process as necessary after completing themicrofiltration process, or any combination thereof.

Embodiment 21: The silk manufacturing system of any one of Embodiments 1to 20 or any combination thereof, wherein the microfiltration modulefurther comprises one or more holding tanks, wherein the tanks may beconfigured to provide additional processing, including one or more ofstoring the solution, temperature control of the solution, or adjustingthe solution concentration to address turbidity or sterility levels.

Embodiment 22: The silk manufacturing system of any one of Embodiments 1to 21 or any combination thereof, wherein the reactor vessel is sized tohave an aspect ratio of height to diameter as defined by a work volumeof about 0.5 to about 5.0.

Embodiment 23: The silk manufacturing system of any one of Embodiments 1to 22 or any combination thereof, wherein the reactor vessel is sized tohave an aspect ratio of height to diameter as defined by a work volumeof about 0.8 to about 2.0.

Embodiment 24: The silk manufacturing system of any one of Embodiments 1to 23 or any combination thereof, wherein the reactor vessel furthercomprises a handling structure for controlling at least one of movementor position of the silk inputs within the vessel.

Embodiment 25: The silk manufacturing system of any one of Embodiments 1to 24 or any combination thereof, further comprising a pre-treatmentsystem configured to condition the silk inputs prior to or atintroduction to the first processing substation.

Embodiment 26: The silk manufacturing system of any one of Embodiments 1to 25 or any combination thereof, wherein the second processingsubstation further comprises a heat exchange system to control atemperature of the silk fibroin-based solution during processing.

Embodiment 27: The silk manufacturing system of any one of Embodiments 1to 26 or any combination thereof, wherein the filtration module isconfigured to remove the second compound from the silk fibroin-basedsolution by circulating the silk fibroin-based solution through thefiltration module until about 5 diavolumes to about at least 8diavolumes are reached.

Embodiment 28: A method of processing silk inputs to obtain silk fibrointhat comprises the steps of introducing a plurality of silk inputs to areactor vessel; introducing a solvent to the reactor vessel; introducinga first compound to the reactor vessel; introducing heat to the reactorvessel contents to promote degumming of the silk inputs; controllingmovement or positioning of the silk inputs within the reactor vessel;rinsing the degummed silk inputs; introducing a second compound to thereactor vessel to dissolve any remaining silk fibroin proteins intosolution; agitating the contents of the reactor vessel; filtering thecontents of the reactor vessel to substantially remove the secondcompound and produce a purified silk fibroin-based solution; andpowderizing the purified silk fibroin-based solution to obtain thepurified silk fibroin in a powder form.

Embodiment 29: A method of processing silk inputs to obtain silk fibrointhat comprises the steps of introducing a plurality of silk inputs to areactor vessel; introducing a solvent to the reactor vessel; introducinga first compound to the reactor vessel; introducing heat to contents ofthe reactor vessel to promote degumming of the cocoons; controllingmovement or positioning of the silk inputs within the reactor vessel;removing at least a portion of the solvent and any degumming residue;rinsing the degummed silk inputs; introducing a second compound to thereactor vessel to dissolve the remaining silk fibroin proteins in tosolution; agitating the contents of the reactor vessel; filtering thecontents of the reactor vessel to substantially remove the secondcompound and produce a purified silk fibroin-based solution; directingthe purified silk fibroin-based solution to a sterilization process toobtain a sterilized silk fibroin-based solution; and powderizing thesterilized silk fibroin-based solution to obtain the silk fibroin in apowder form.

Embodiment 30: A method of processing silk inputs to obtain food gradesilk fibroin that comprises the steps of providing a reactor vesselconfigured to extract silk fibroin proteins via degumming, rinsing, anddissolving processes therein, wherein the vessel comprises at least oneinlet port, at least one outlet port; and a liquid jacket configured toprovide heat exchange with the vessel and its contents; introducing aplurality of silk inputs to the reactor vessel via the at least oneinlet port; introducing a solvent to the reactor vessel via the at leastone inlet port; introducing a first compound to the reactor vessel viathe at least one inlet port; heating the contents of the reactor vesselvia the liquid jacket to a temperature of about 50° C. to about 150° C.to promote degumming of the silk inputs; controlling movement orpositioning of the silk inputs within the reactor vessel; removing atleast a portion of the solvent and any degumming residue via the atleast one outlet port; rinsing the degummed silk inputs; introducing asecond compound to the reactor vessel via the at least one inlet port todissolve the remaining silk fibroin proteins to form a silkfibroin-based solution; agitating the contents of the reactor vessel;outputting the silk fibroin-based solution including the second compoundto a filtration module via the at least one outlet port; filtering thesilk fibroin-based solution including the second compound tosubstantially remove the second compound and produce a purified silkfibroin-based solution, wherein the filtration module is configured toremove the second compound from the silk fibroin-based solution bycirculating the silk fibroin-based solution through the filtrationmodule until about 1 diavolume to about at least 12 diavolumes arereached; and powderizing the purified silk fibroin-based solution via aspray dryer to obtain the silk fibroin in a powder form such that thewater activity level of the powder is less than 0.9.

Embodiment 31: A method of processing silk inputs to obtain food gradesilk fibroin that comprises the steps of providing a reactor vesselconfigured to extract silk fibroin proteins via degumming, rinsing, anddissolving processes therein, wherein the vessel comprises at least oneinlet port, at least one outlet port; and a liquid jacket configured toprovide heat exchange with the vessel and its contents; introducing aplurality of silk inputs to the reactor vessel via the at least oneinlet port; introducing a solvent to the reactor vessel via the at leastone inlet port; introducing a first compound to the reactor vessel viathe at least one inlet port; heating the contents of the reactor vesselvia the liquid jacket to a temperature of about 50° C. to about 150° C.to promote degumming of the silk inputs; controlling movement orpositioning of the silk inputs within the reactor vessel; removing atleast a portion of the solvent and any degumming residue via the atleast one outlet port; rinsing the degummed silk inputs such that thesilk inputs are substantially free of sericin; introducing a secondcompound to the reactor vessel via the at least one inlet port todissolve the remaining silk fibroin proteins to form a silkfibroin-based solution; agitating the contents of the reactor vessel;sterilizing the silk fibroin-based solution to obtain a sterilized silkfibroin-based solution; outputting the sterilized silk fibroin-basedsolution including the second compound to a filtration module via the atleast one outlet port; filtering the silk fibroin-based solutionincluding the second compound to substantially remove the secondcompound and produce a purified silk fibroin-based solution, wherein thefiltration module is configured to remove the second compound from thesilk fibroin-based solution by circulating the silk fibroin-basedsolution through the filtration module until about 1 diavolume to aboutat least 12 diavolumes are reached, wherein the purified silkfibroin-based solution comprises less than about 650 parts per million(ppm) of one or more salts or non-organic particulates; and powderizingthe purified silk fibroin-based solution via a spray dryer to obtain thesilk fibroin in a powder form such that the water activity level of thepowder is less than 0.9.

Embodiment 32: The silk manufacturing system of any one of Embodiments28 to 31 or any combination thereof, wherein the silk inputs come from aBombyx mori silkworm.

Embodiment 33: The silk manufacturing system of any one of Embodiments28 to 32 or any combination thereof, wherein a packing density of thesilk inputs in the reactor vessel is between about 1% and about 70%.

Embodiment 34: The silk manufacturing system of any one of Embodiments28 to 33 or any combination thereof, wherein a packing density of thesilk inputs in the reactor vessel is greater than 5%.

Embodiment 35: The silk manufacturing system of any one of Embodiments28 to 34 or any combination thereof, wherein a packing density of thesilk inputs in the reactor vessel is greater than 15%.

Embodiment 36: The silk manufacturing system of any one of Embodiments28 to 35 or any combination thereof, wherein a packing density of thesilk inputs in the reactor vessel is greater than 25%.

Embodiment 37: The silk manufacturing system of any one of Embodiments28 to 36 or any combination thereof, wherein the filtering stepcomprises purifying the silk fibroin-based solution via diafiltration.

Embodiment 38: The silk manufacturing system of any one of Embodiments28 to 37 or any combination thereof, wherein the filtering stepcomprises purifying the silk fibroin-based solution via tangential flowfiltration.

Embodiment 39: The silk manufacturing system of any one of Embodiments28 to 38 or any combination thereof, wherein the method furthercomprises the step of performing a sterilization process to obtain afood grade quality silk fibroin-based solution prior to the powderizingstep.

Embodiment 40: The silk manufacturing system of any one of Embodiments28 to 39 or any combination thereof, wherein the sterilization processcomprises the step of directing the purified silk fibroin-based solutionto a microfiltration module.

Embodiment 41: The silk manufacturing system of any one of Embodiments28 to 40 or any combination thereof, wherein the step of directing thepurified silk fibroin-based solution to a microfiltration modulecomprises directing the purified silk fibroin-based solution through afirst microfiltration stage having a pore size between about 0.7 µm andabout 5 µm and directing the purified silk fibroin-based solutionthrough a second microfiltration stage having a pore size between about0.05 µm and about 0.8 µm.

Embodiment 42: The silk manufacturing system of any one of Embodiments28 to 41 or any combination thereof, further comprising the step ofadjusting a temperature of the silk fibroin-based solution duringprocessing.

Embodiment 43: The silk manufacturing system of any one of Embodiments28 to 42 or any combination thereof, further comprising apost-powderization step comprising at least one of: agglomerating thesilk fibroin powder, conditioning the silk fibroin powder, testing thesilk fibroin powder, or packaging the silk fibroin powder into, forexample, a food-safe container.

Embodiment 44: The silk manufacturing system of any one of Embodiments28 to 43 or any combination thereof, wherein the filtering stepcomprises utilizing at least one spiral wound membrane.

Embodiment 45: The silk manufacturing system of any one of Embodiments28 to 44 or any combination thereof, wherein the at least one inlet portcomprises a first inlet port configured to receive the silk inputs, thefirst compound, and the second compound and a second inlet portconfigured to receive the solvent; and the at least one outlet portcomprises a first outlet port configured to output the silkfibroin-based solution and a second outlet port configured to output atleast a portion of the solvent and any degumming residue.

Still other aspects, embodiments, and advantages of these exemplaryaspects and embodiments, are discussed in detail below. Moreover, it isto be understood that both the foregoing information and the followingdetailed description are merely illustrative examples of various aspectsand embodiments, and are intended to provide an overview or frameworkfor understanding the nature and character of the claimed aspects andembodiments. Accordingly, these and other objects, along with advantagesand features of the present disclosure herein disclosed, will becomeapparent through reference to the following description and theaccompanying drawings. Furthermore, it is to be understood that thefeatures of the various embodiments described herein are not mutuallyexclusive and can exist in various combinations and permutations.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the disclosure and are not intended as adefinition of the limits of the disclosure. For purposes of clarity, notevery component may be labeled in every drawing. In the followingdescription, various embodiments of the present disclosure are describedwith reference to the following drawings, in which:

FIG. 1 shows an example of a silk manufacturing process in accordancewith one or more embodiments of the disclosure;

FIG. 2 shows an example of a first processing substation for use in asilk manufacturing process in accordance with one or more embodiments ofthe disclosure;

FIG. 3 shows an example of a second processing substation for use in asilk manufacturing process in accordance with one or more embodiments ofthe disclosure;

FIG. 4 shows an example of auxiliary equipment for integration with asilk manufacturing process in accordance with one or more embodiments ofthe disclosure;

FIGS. 5A-5E show examples of various third processing substations asintegrated in silk manufacturing processes in accordance with one ormore embodiments of the disclosure;

FIG. 6A shows one example of a fourth processing substation for use in asilk manufacturing process in accordance with one or more embodiments ofthe disclosure;

FIG. 6B shows another example of a fourth processing substation for usein a silk manufacturing process in accordance with one or moreembodiments of the disclosure;

FIGS. 7A-7D show alternative examples of silk manufacturing processes inaccordance with one or more embodiments of the disclosure;

FIG. 8 shows yet another example of a silk manufacturing process inaccordance with one or more embodiments of the disclosure;

FIGS. 9A-9C show examples of screens for integration with a silkmanufacturing process in accordance with one or more embodiments of thedisclosure;

FIG. 10 shows an example of screen placement in a first processingsubstation in accordance with one or more embodiments of the disclosure;

FIG. 11 shows another example of screen placement in a first processingsubstation in accordance with one or more embodiments of the disclosure;

FIG. 12 shows an exemplary graph of the molecular weights (MW) of silkfibroin fragments in an exemplary silk film and/or coating; and

FIG. 13 shows an exemplary graph of the molecular weights (MW) of silkfibroin fragments in an exemplary silk film and/or coating.

DETAILED DESCRIPTION

Some implementations of the present disclosure will now be describedmore fully hereinafter with reference to the accompanying figures, inwhich some, but not all implementations of the disclosure are shown.Indeed, various implementations of the disclosure may be embodied inmany different forms and should not be construed as limited to theimplementations set forth herein; rather, these example implementationsare provided so that this disclosure will be thorough and complete, andwill fully convey the scope of the disclosure to those skilled in theart.

Unless specified otherwise or clear from context, references to first,second, third or the like should not be construed to imply a particularorder. A feature described as being above another feature (unlessspecified otherwise or clear from context) may instead be below, andvice versa; and similarly, features described as being to the left ofanother feature may instead be to the right, and vice versa. Also, whilereference may be made herein to quantitative measures, values, geometricrelationships or the like, unless otherwise stated, any one or more ifnot all of these may be absolute or approximate to account foracceptable variations that may occur, such as those due to engineeringtolerances or the like.

The disclosure relates to systems and methods for improving themanufacturing of silk fibroin-based solutions containing silk fibroinfrom silk cocoons.

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout theSpecification.

As used in this Specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive and covers both “or” and “and”.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include A and B; A or B; A (alone);and B (alone). Likewise, the term “and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the followingembodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; Aand B; B and C; A (alone); B (alone); and C (alone).

The terms “e.g.,” and “i.e.” as used herein, are used merely by way ofexample, without limitation intended, and should not be construed asreferring only those items explicitly enumerated in the specification.

The terms “or more”, “at least”, “more than”, and the like, e.g., “atleast one” are understood to include but not be limited to at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 ormore than the stated value. Also included is any greater number orfraction in between.

Conversely, the term “no more than” includes each value less than thestated value. In one embodiment, “no more than 100” includes 100, 99,98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81,80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63,62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, and 0. Also included is any lesser number orfraction in between.

The terms “plurality”, “at least two”, “two or more”, “at least second”,and the like, are understood to include but not limited to at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73, 75, 76, 81, 84, 89,92, 71, 72, 74, 77, 78, 79, 80, 82, 83, 85, 86, 87, 88, 90, 91, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more.Also included is any greater number or fraction in between.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. It is understood thatwherever embodiments are described herein with the language“comprising,” otherwise analogous embodiments described in terms of“consisting of” and/or “consisting essentially of” are also provided.The term “consisting of” excludes any element, step, or ingredient notspecified in the claim. In one embodiment, “consisting of” is defined as“closing the claim to the inclusion of materials other than thoserecited except for impurities ordinarily associated therewith. A claimwhich depends from a claim which “consists of” the recited elements orsteps cannot add an element or step. The terms “consisting essentiallyof” or “consists essentially” likewise has the meaning ascribed in U.S.Patent law and the term is open-ended, allowing for the presence of morethan that which is recited so long as basic or novel characteristics ofthat which is recited is not changed by the presence of more than thatwhich is recited, but excludes prior art embodiments.

Unless specifically stated or evident from context, as used herein, theterm “about” refers to a value or composition that is within anacceptable error range for the particular value or composition asdetermined by one of ordinary skill in the art, which will depend inpart on how the value or composition is measured or determined, i.e.,the limitations of the measurement system. In one embodiment, “about” or“approximately” may mean within one or more than one standard deviationper the practice in the art. “About” or “approximately” may mean a rangeof up to 10% (i.e., ±10%). Thus, “about” may be understood to be within10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or0.001% greater or less than the stated value. In one embodiment, about 5mg may include any amount between 4.5 mg and 5.5 mg. Furthermore,particularly with respect to biological systems or processes, the termsmay mean up to an order of magnitude or up to 5-fold of a value. Whenparticular values or compositions are provided in the instantdisclosure, unless otherwise stated, the meaning of “about” or“approximately” should be assumed to be within an acceptable error rangefor that particular value or composition.

Further, as used in the following, the terms “preferably”, “morepreferably”, “most preferably”, “particularly”, “more particularly”,“specifically”, “more specifically” or similar terms are used inconjunction with optional features, without restricting furtherpossibilities. Thus, features introduced by these terms are optionalfeatures and are not intended to restrict the scope of the claims in anyway. The disclosure may, as the skilled person will recognize, beperformed by using alternative features. Similarly, features introducedby “in an embodiment of the disclosure” or similar expressions areintended to be optional features, without any restriction regardingfurther embodiments of the disclosure, without any restrictionsregarding the scope of the disclosure, and without any restrictionregarding the possibility of combining the features introduced in suchway with other optional or non-optional features of the disclosure.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to be inclusive of the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Units, prefixes, and symbols used herein are provided using theirSystème International de Unites (SI) accepted form. Numerical ranges areinclusive of the numbers defining the range. Additionally, wheremultiples of the same components are described, the multiples may bereferred to individually (e.g., ##a, ##b, ##c, etc.) or collectively(##).

DESCRIPTION

FIG. 1 depicts a process 100 for manufacturing a silk fibroin-basedsolution and obtaining silk-fibroin in a powder form. Specifically, theprocess 100 includes performing a degumming process (step 120) forextracting silk fibroin from silkworm cocoons 102 and then a dissolutionprocess on the degummed silk fibroin (step 140) where the silk fibroinis dissolved in a heated chaotropic agent solution. Next, the process100 includes exposing the fibroin-based solution to a purificationprocess (step 160) where the chaotropic agent is removed from thedissolved silk fibroin solution, and finally, the silk fibroin solutionis dried to obtain the silk fibroin (e.g., via powderization) (step180). The quality of the silk fibroin obtained may be improved byimproving the quality of the silk fibroin-based solution.

During the manufacturing process of silk fibroin solution, a process canbe used to reduce turbidity and kill microbes to obtain a silk fibroinsolution that contains the desired performance and safety requirements.Excess turbidity is undesirable in the silk fibroin solution because itmay impact the tackiness of a coating made from the silk fibroin-basedsolution, hinder the barrier forming properties of the silkfibroin-based solution, and may cause a coating formed from the silkfibroin-based solution to look cloudy or milky. For this reason,turbidity should be kept under about 0.800 optical density measured at awavelength of 600 nm (OD660). Accordingly, methods to meet theserequirements are desirable and may include, for example, the integrationof a sterilization step/substation (see 1110 in FIG. 5A and 610 a/b inFIG. 7 ) as described herein.

Generally, the various systems and substations described herein may beinterconnected via conventional plumbing techniques and may include anynumber and combination of components, such as pumps, valves, sensors,gauges, etc., to monitor and control the operation of the varioussystems and processes described herein, either manually orautomatically. The various components made from materials suitable forthe temperatures and materials to which they are exposed and may be usedin conjunction with a controller as described herein.

FIG. 2 depicts a first substation 220 configured for receiving thesilkworm cocoons 202 and performing degumming, dissolving and rinsingoperations on the silkworm cocoons to obtain a silk fibroin-basedsolution, Ideally the silk fibroin-based solution would be substantiallyfree of sericin after the degumming process. As shown, the substation220 includes a reactor vessel 222 having a first inlet port 226 aconfigured to receive the silkworm cocoons 202 and one or moreingredients 232 (e.g., soda ash, a chaotropic agent), a second inletport 226 b configured to receive a solvent (e.g., water), and at leastone outlet configured to output the silk fibroin-based solution. Thereactor vessel 222 is configured to process the silkworm cocoons into asilk fibroin solution by at least one of degumming, rinsing, anddissolving within the single glass-lined vessel. In alternativearrangements, one or more first substations generally, and one or morereactor vessels specifically, may be provided to suit a particularapplication. See, for example, FIGS. 7A-7C. In some examples, multiplesmaller vessels may be used to optimize the process by, for example,making the heating and cooling of the solution more efficient. In someembodiments, the substation 220 includes a heat exchanger 242 forconditioning the solvent prior to introduction into the vessel 222.

The first processing substation 220 also includes a water or oil jacket224 disposed about the reactor vessel 222 that is configured to provideheat exchange (e.g., heating or cooling as necessary) with the vessel222 and its contents. The water or oil jacket 224 includes a heatexchange circuit 236 that includes a pump 237 for recirculating aheating/cooling medium in fluid communication with, for example, twoheat exchangers 239 a, 239 b that are in fluid communication with one ormore of steam or cooling liquid as necessary to control the temperatureof the contents of the vessel 222. The first processing substation 220also has the ability to pressurize its contents. The first processingsubstation is configured to pressurize the contents to a pounds persquare inch (psi) of about 0 psi to about 20 psi, from about 0 to about10 psi, from about 0 to about 5 psi, from about 0.1 to about 20 psi,from about 0.1 psi to about 10 psi, and from about 0.1 psi to about 5psi. The pressure can be applied during any of the steps to obtain asilk fibroin-based solution, including degumming, rinsing, anddissolving.

The first processing substation 220 also includes a plurality of inputsand outputs 238, 244 for introducing and/or removing a solvent, steam,cooling water, condensate, etc. to, for example, the vessel 222 and/orthe water or oil jacket 224 via their corresponding inlets/outlets. Forexample, in some embodiments, input 238 a. is configured to introducesoftened water 245 to the heat exchanger 242 and then to the vessel 222via the inlet 226 b, inputs 238 b, 238 c introduce steam to the heatexchangers 242 and 239 b respectively, and input 238 d introducescooling water to one of the heat exchangers 239 a. The outputs 244 a,244 b, 244 c are configured to remove the condensate and cooling waterfrom the heat exchangers 239, 242. In other embodiments oil can besubstituted for water to achieve the same cooling or heatingrequirements.

The first processing substation 220 may further include equipment 234configured to agitate the contents of the reactor vessel 222, such as,for example, a mixer, a vibration plate, a magnetic stirrer, sonicator,liquid jet streams, air streams, etc. In various embodiments, theagitation equipment 234 may be disposed proximate a bottom surface ofthe reactor vessel 222. In some embodiments, the agitation equipment 234is a mixer having a unitary shaft and impeller 235. The impeller 235 maybe configured for axial flow, radial flow, and/or tangential flow, andmay be run in reverse. Additionally, the impeller 235 may be coated witha substance to resist attachment of silk fibers and/or have a surfacefinish of the blades (e.g., a surface roughness below some thresholdvalue). The mixer may have interchangeable impellers, where theimpellers may be configured to suit particular processes and have one ormore of flat blades, curved blades, pitched blades, finger blades,anchor blades, gate blades, ribbon blades, etc. having different shapes,pitch, etc. In some embodiments, the impeller assembly includes aslidable sleeve that may be configured to compress the cocoons and/orremove build-up on the impeller (e.g., push or scrap the cocoons off ofthe impeller).

In further embodiments, the reactor vessel 222 includes a second outlet228 b for removing at least a portion of the solvent and any residuetherein (e.g., dissolved sericin), which can be sent to waste,recirculated, or recycled. The reactor vessel 222 may be sized to havean aspect ratio of height to diameter as defined by a work volume. Thevolume of the vessel will vary to suit a particular application (e.g.,finished yields) and may range from about 0.2 liters to about 150,000liters, preferably about 0.5 liters to about 5,000 liters. The vesselcontents may include a plurality of silkworm cocoons 202 (with orwithout pre-treatment), a solvent 244 a (e.g., water), and a compound.The water or oil jacket 224 is configured to heat the contents to atemperature of about 50° C. to about 150° C., preferably about 85° C. toabout 125° C. Generally, the process times, temperatures, pH, and othersolution characteristics may vary to suit a particular application, suchas the type of silk source.

The reactor vessel 222 may also include a handling structure orequipment 230 configured to control the movement and/or the position ofthe silkworm cocoons 202 within the vessel 222 (e.g., prevent floatingof the cocoons). The equipment 230 may include, for example, a screen ornetting disposed proximate a lower portion of the vessel 222 andconfigured to separate the silkworm cocoons from the agitation equipment234, a chute or funnel structure in communication with the first inletand configured to direct the silkworm cocoons to a particular locationwithin the vessel 222 during introduction thereof, a recirculationsystem configured to draw a portion of the solution from a lower portionof the vessel 222 and reintroduce the solution to an upper portion ofthe vessel 222 and/or introduce fresh water to push the silkworm cocoonsdown into the solution, a vertically moveable sieve (e.g., a perforatedplunger or a vented, floating lid) disposed within the vessel andconfigured to “push” any solids within the solution towards a lowerportion of the vessel, one or more spray balls, and one or more bafflesdisposed within the vessel and extending from an inner wall thereof,where the baffles direct the movement of the solution and contentstherein. In one embodiment, the equipment 230 includes one or more cagesor nets disposed within the vessel 222 to ensure that the silkwormcocoons are spaced throughout the vessel 222. For example, the silkwormcocoons may be separated into a plurality of spherical or cubical cages.

The first processing substation 220, and system generally, may includeone or more valve assemblies 225, inlets 226, and/or outlets 228 (withmanual or automatic actuators) that are configured to control theintroduction to and removal from the first processing substation and/orthe reactor vessel 222 of any component, such as, for example, silkwormcocoons, compounds, solvents, waste solutions, residues, steam, coolingwater, and final silk fibroin-based solutions. The first processingsubstation 220, and system generally, may include at least one sensor227 configured to sense one or more of solution temperatures,concentrations, flow rates, pH, fluid levels, turbidity, particle size,molecular weight, pressure, etc., which may be used to control (with orwithout human intervention) the operation of the various processes. Oncethe desired silk fibroin-based solution has been obtained, which may bedetermined manually or via one or more sensed characteristics, thesolution is directed (e.g., via pumps, valves, etc. as needed) to thenext substation as described below. In various embodiments, the systemsdescribed herein may include a clean-in-place (CIP) module 246 (e.g., amobile cart) that can be fluidly coupled to the substations to performmaintenance thereon.

FIG. 3 depicts a second substation 260 configured for receiving the silkfibroin-based solution and filtering the solution to remove thechaotropic agent from the solution. As shown, the substation 260includes a holding vessel 268 and a filtration module 266 housing atleast one membrane. The substation 260 includes at least one input 261configured to receive the silk fibroin-based solution including thechaotropic agent from the first substation 220 and at least one outlet263 configured to output the purified silk fibroin-based solution with areduced chaotropic agent concentration (i.e., the retentate 270, 270′)and another outlet 274 configured to output a waste stream, such as thepermeate 272 from the filtration module 266. The silk fibroin-basedsolution including the chaotropic agent is introduced to the vessel 268via an inlet 267 a disposed thereon.

Generally, the filtration module 266 is configured to remove thechaotropic agent from the silk fibroin-based solution via diafiltration.In some cases, the flow through the module is tangential to a surface ofthe membrane. The silk fibroin-based solution may also experience somelevel of concentration that may be beneficial in later operations. Thesilk fibroin-based solution may be circulated through the filtrationmodule for a duration defined by about 1 diavolumes to about at least 12diavolumes, preferably about 3 diavolumes to about 10 diavolumes, andmore preferably about 5 diavolumes to about 9 diavolumes. In some cases,the concentrations levels of the chaotropic agent in the retentateand/or the pressure drop across the filtration module may also bemonitored to determine a state of the process. The filtration module 266may include any number and type of membranes to suit a particularapplication. In one embodiment, the module 266 includes one or morespiral wound membranes, which may be provided in multiple stages. Forexample, the silk fibroin-based solution may pass through the filtrationmodule 266, and the various stages thereof, in series, parallel, or bothto suit a particular application.

The holding vessel 268 may include one or more inlets 267 b, 267 cconfigured to introduce a rinse solution, such as a softened water(input 264 a) or a reverse osmosis water (input 264 b) to the silkfibroin-based solution during the filtration process. The vessel 268further includes an outlet 265 for removing the solution 270 anddirecting the solution to the filtration module 266 via a pumping system276. The at least partially purified solution 270′ is directed back tothe holding vessel 268 (via inlet 267 d), where it may be exposed toadditional rinsing and circulation through the filtration module 266.The permeate 272 may be output to waste (with or without furtherprocessing) or recycled if feasible. Once the solution 270, 270′ hasreached a desired level of purification, as determined manually orautomatically, the solution 270, 270′ is output to another processingsubstation (e.g., sterilization) via a valve arrangement 225. In someembodiments, the purified solution may be removed from the secondprocessing system 260 via an alternative line 269 and valve assembly 225located downstream of the filtration module 266. In some embodiments,the solution 270, 270′ is output directly to the fourth processingsubstation 280, 380 rather than another substation through the outlet263.

The second processing substation 260 may also include a heat exchangesystem 262, including any valves, pumps, controls, etc. as needed tocontrol a temperature of the silk fibroin-based solution duringprocessing. As shown in FIG. 3 , the heat exchange circuit 262 isdisposed in the return line to control the temperature of the at leastpartially purified solution 270′ exiting the filtration module 266;however, the heat exchange circuit 262 may be located elsewhere to suita particular application. The circuit 262 further includes ports 271 a,271 b for introducing and/or removing a cooling (or heating) medium.

In various embodiments of the systems disclosed herein, an optionalpre-filtration substation 250 may be disposed between the firstprocessing substation 220 and the second processing substation 260. Thepre-filtration substation 250 shown in FIG. 4 may include a transferpump 252 to assist with the transfer of the silk fibroin-based solutionto the second processing substation 260, one or more filtration modules254 to suit a particular application, and a heat exchange circuit 256.In some embodiments, the pre-filtration substation includes a valveassembly 225 that may be configured to bleed off a portion of the silkfibroin-based solution that may contain an excessive amount of acontaminant (e.g., sericin) to waste, with or without cooling as needed.

FIGS. 5A-5E are exemplary implementations of various third processingsubstations 1110, 1210, 1310, 1410, 1510 for sterilization that may beincorporated into the overall production process for a silkfibroin-based solution containing silk fibroin. These figures do notshow all the possible implementations of the systems and processes andare generally shown in relationship to the second processing substations1160, 1260, 1360, 1460, 1560.

Generally, one of the major concerns when processing the silkfibroin-based solution is that the process does not negatively impactthe silk fibroin-based solution, including the silk fibroin, or itsperformance. For example, using a filter with a pore size that is toosmall could damage the shear-sensitive silk fibroin in the silkfibroin-based solution, which could reduce the barrier formingproperties of the silk fibroin-based solution. In another example, afilter could remove some of the silk fibroin from the solution, alteringthe molecular weight (Mw) of the silk fibroin-based solution, forexample, rendering the Mw too high or too low, or by narrowing thepolydispersity index (PDI). As another example, the microfiltration stepcould reduce the volume of the silk fibroin-based solution, which shouldbe limited. The goal of the filtration step is to provide a process thatcan meet all the requirements described herein, without negativelyimpacting the performance of the silk fibroin-based solution.

One option to obtain these results is through the sterilization processof the third processing substations 1110, 1210, 1310, 1410, 1510described herein. The third processing substations depicted herein usemicrofiltration to addresses these concerns and are capable of producinga silk fibroin-based solution that falls under the allowable limits foreach. Additional sterilization systems and processes are disclosed inU.S. Provisional Pat. application no. 63/191,441, filed May 21, 2021,which is hereby incorporated by reference herein in its entirety. Themicrofiltration process could entail utilizing multiple, different typesof filters, (e.g., spiral, membrane, cartridge, hollow fiber, plate andframe, cartridges with O-rings), materials, membrane structures, poresizes, etc.), different transmembrane pressures, and/or the number andconfigurations of the filters (e.g., two or more filter stages arrangedin a series configuration, where each filter stage may incorporate morethan one filter/membrane in different configurations). Generally, theexact number and arrangement of filter stages and/or the filtersincluded therein, along with membrane pore sizes, may vary to suit aparticular application; for example, to accommodate different flowrates, volumes, target pressure drops, target turbidity levels,sterility levels, solvents used, etc.

FIG. 5A shows an example of the sterilization process/third processingsubstation 1110 incorporated with the purification process/secondprocessing substation 1160, where the third substation 1110 is disposeddownstream of the second substation 1160 and includes the use of twopumps 1114, two filter stages 1116, and two holding tanks 1118. Asshown, the path of the silk fibroin-based solution after it leaves thepurification process of the second substation 1160 is directed to afirst pump 1114 a, which passes the solution to and through a firstfilter stage 1116 a and into a first holding tank 1118 a. A second pump1114 b in fluid communication with the first holding tank 1118 atransfers the silk fibroin-based solution to and through a second filterstage 1116 b and into a second holding tank 1118 b. The silkfibroin-based solution may be directed to another process as necessaryafter completing the microfiltration process of the third substation1110.

FIG. 5B shows another example of a sterilization process/thirdprocessing substation 1210, where the purification process 1260 stillprecedes the sterilization process/third processing substation 1210, butincludes the use of one pump 1214, two filter stages 1216, and oneholding tank 1218. As shown, the path of the silk fibroin-based solutionafter it leaves the purification process of the second substation 1260is directed to the single pump 1214, which passes the silk fibroin-basedsolution to and through the first filter stage 1216 a and then to andthrough the second filter stage 1216 b and into the holding tank 1218.Again, the silk fibroin-based solution may be directed to anotherprocess as necessary after completing the sterilization process,including, for example, back through the third processing substation1210 for a second pass, quality testing, or to powderization.

FIG. 5C shows yet another example of a sterilization process/thirdprocessing substation 1310, but where the sterilization process isincorporated with the purification process/second processing substation1360 and includes two pumps 1314, two filter stages 1316, and oneholding tank 1318. As shown, the path of the silk fibroin-based solutionis introduced to the first filter stage 1316a via the first pump 1314aand then to the purification process 1360. The silk fibroin-basedsolution exiting the purification process 1360 is directed to the secondpump 1314 b, which passes the purified silk fibroin-based solution toand through the second filter stage 1316 b and into the holding tank1318. In this embodiment, the sterilization process occurs both beforeand after the purification process and includes the use of two pumps,two filters, and one holding tank.

FIG. 5D shows still another example of a sterilization process/thirdprocessing substation 1410, where the sterilization process is carriedout prior to the purification process 1460 and includes one pump 1414,two filter stages 1416, and one holding tank 1418. As shown, the path ofthe silk fibroin-based solution is introduced to the first and secondfilter stages 1416 a, 1416 b via the pump 1414 and then directed to thepurification process 1460. The silk fibroin-based solution exiting thepurification process 1460 is directed to the holding tank 1418. In thisembodiment, the sterilization process 1410 occurs before thepurification process 1460 and includes the use of one pump, two filters,and one holding tank; however, other quantities of pumps, filter stages,and tanks may be incorporated to suit a particular application.

FIG. 5E shows another example of a sterilization process/thirdprocessing substation 1510 similar to some of those described above andwhere the purification process/second processing substation 1560precedes the sterilization process 1510 and includes the use of twopumps 1514, two filter stages 1516, one holding tank 1518, and oneauxiliary piece of equipment 1512. As shown, the path of the silkfibroin-based solution after it leaves the purification process 1560 isdirected to the first pump 1514 a, which first passes the solution toand through a heat exchange module 1512 that may be used to heat and/orcool the silk fibroin-based solution prior to its introduction to thefirst filter stage 1516 a. However, the heat exchange module 1512 couldbe located after the first or second filter stage 1516 a, 1516 b and/orbefore the holding tank 1518. The second pump 1514 b is in fluidcommunication with the first filter stage 1516 a and transfers the silkfibroin-based solution to and through a second filter stage 1516 b andinto the holding tank 1518.

In other embodiments, different numbers and configurations (e.g., seriesor parallel) of filter stages may be used. Multiple tanks or pumps mayalso be used to obtain the desired throughput of the filters and properpressure to achieve optimal filtration. Additionally, the tanks mayinclude structure for further treating the silk fibroin-based solutionto further reduce turbidity and/or microbes, such as, for exampleadjusting the solution composition.

FIGS. 6A and 6B depict alternative fourth processing substations 280,380 for powderizing the sterilized silk fibroin-based solution. As shownin FIG. 6A, the fourth substation 280 includes a dryer feed mixer vessel282 and a spray dryer 288 in fluid communication with a plurality ofinputs and outputs 284 (e.g., softened water 284 a, compressed air 284b, exhaust 284 c, chilled water in 284 d, and chilled water out 284 e).Generally, the fourth processing substation 280 includes an input 281configured to receive the purified and/or sterilized silk fibroin-basedsolution and an output 283 configured to output a silk-fibroin powderthat is easily instantizable. In some embodiments, the water activitylevel of the silk-fibroin powder may be from about 0.01 to about 1.0,preferably under 0.85. The dryer feed mixer vessel 282 holds the silkfibroin-based solution prior to drying and may be configured to treatthe silk fibroin-based solution prior to drying to, for example, enhancepowderization or produce a more instantizable powder. The silkfibroin-based solution is transferred from the vessel 282 to the spraydryer 288 via a pumping system 286. In some embodiments, silkfibroin-based solution from different batches with different molecularweight profiles (e.g., lower molecular weight silk fibroin may be addedto higher molecular weight silk fibroin) may be mixed together in thefeed tank prior to powderization. In some embodiments, an additive couldbe added to the feed tank prior to powderization. These additives can beany of those known to one of ordinary skill in the art, includingkosmotropic components, humectants, anticaking agents, antifoamingagents, oils, sugars, desiccants, catalysts, or any of those listed inU.S. Pat. Publication No. 2020-0178576 A1, which is incorporated hereinby reference.

The fourth processing substation 380 depicted in FIG. 6B issubstantially identical to the substation 280 of FIG. 6A, insofar as thesubstation 380 includes a dryer feed mixer vessel 382 and a spray dryer388 in fluid communication with a plurality of inputs and outputs 384and configured to receive the purified and/or sterilized silkfibroin-based solution via an input 381 and output a silk-fibroin powdervia an output 383. The fourth processing substation 380 includesadditional equipment disposed downstream of, or incorporated with, thespray dryer 388. Specifically, the substation 380 includes equipment 391for providing an additive(s) to the silk fibroin powder or otherwiseconditioning the powder for, for example, enhanced performance. In oneembodiment, the equipment 391 is agglomeration equipment, such as anexternal fluid bed or a fluid bed integrated with the powderizationequipment. The agglomeration equipment may aid in agglomeration of thepowdered silk fibroin protein, which may improve dispersibility,instantization, or wettability properties of the powdered silk fibroinprotein. Any suitable agglomeration equipment may be utilized. Thesubstation also includes packaging equipment 393 for appropriatelypackaging the silk fibroin powder.

FIGS. 7 and 8 depict alternative examples of systems and processes formanufacturing silk-fibroin solutions and obtaining a silk fibroin powdertherefrom.

Generally, FIGS. 7A-7D depict alternative systems/processes 600, 600′,600″, 600‴ for manufacturing a silk fibroin solution. The system 600 ofFIG. 7A includes an optional pre-treatment substation 605 forpre-treating the silkworm cocoons (e.g., shredding, soaking, pupaeremoval (e.g., sieve, vibrating screen, etc.), etc.) and a firstprocessing substation 620 downstream thereof for performing a degumming,rinsing, and dissolution process to obtain a silk fibroin-basedsolution, with an optional heat exchange substation 612 a forconditioning the solution. In some embodiments, the pre-treatmentsubstation 605 may include a continuous soaking process where thesilkworm cocoons are soaked in a heated solution (e.g., water comprisingthe first compound) and then fed to the reactor vessel, with or withoutdewatering. In addition, during processing in the first processingsubstation 620, the reactor vessel may be drained, refilled, andreheated at various stages of the process (e.g., at the halfway point)and/or multiple degumming processes carried out.

Disposed downstream of the first substation 620 and in fluidcommunication therewith is a first sterilization module 610 a of a thirdprocessing substation for treating the silk fibroin-based solution priorto purifying the solution at the second processing substation 660. Thesecond processing substation 660 may include an optional heat exchangesubstation 612 b. Next, the solution is directed to a secondsterilization module 610 b of the third processing substation, and thenthe sterilized silk fibroin-based solution is transferred to a fourthprocessing substation 680 for powderizing the silk fibroin-basedsolution. The silk fibroin powder may then be directed to an optionalpost-treatment substation 615 for additional processing and/orpackaging. The systems and processes described herein may includeadditional or different processing substations as necessary to suit aparticular application.

The systems 600′, 600″ of FIGS. 7B and 7C are similar to the system 600described above insofar as they include an optional pre-treatmentsubstation 605′, 605″, one or more first processing substations 620′,620″, one or more sterilization modules 610′, 610″ (i.e., thirdprocessing substation), a second processing substation 660′, 660″ forpurifying the solution, a fourth processing substation 680′, 680″ forpowderizing the silk fibroin-based solution, and an optionalpost-treatment substation 615′, 615″. Specifically, the system 600′depicted in FIG. 7B incorporates multiple first processing substations(or multiple DRD vessels) 620 a′, 620 b′, 620 c′ arranged in parallel.For example, multiple smaller substations may be used in parallel tospeed up production and/or accommodate a particular plant footprint. Thesystem 600″ depicted in FIG. 7C also includes multiple first processingsubstations (or multiple DRD vessels) 620 a″, 620 b″, but arranged inseries. In some embodiments, a system 600‴ as shown in FIG. 7D is used.The system 600‴ includes one or more first processing substations 620‴for performing a degumming, rinsing, and dissolution process to obtain asilk fibroin-based solution, one or more second processing substations660‴ for purifying the solution, and one or more fourth processingsubstations 680‴ for powderizing the silk fibroin-based solution.Generally, the specific number and arrangement of the first processingsubstations 620, 620′, 620″, 620‴ may vary to suit a particularapplication.

FIG. 8 depicts a system/process 700 for manufacturing a silk fibroinsolution that includes a first processing substation 720, a secondprocessing substation 760 disposed downstream of the first substation, athird processing substation 710 disposed downstream of the secondsubstation, and a fourth processing substation 780 disposed downstreamof the third substation. The various substations are similar to thosedescribed herein, insofar as the first substation 720 is configured toreceive a plurality of cocoons, a solvent, and one or more compounds forprocessing to obtain a silk fibroin-based solution; the secondsubstation is configured to filter the silk fibroin-based solution tosubstantially remove one or more compounds and produce a purified silkfibroin-based solution; the third substation is configured to receivethe purified silk fibroin-based solution and sterilize same to obtain a“food grade” quality silk fibroin-based solution; and the fourthsubstation is configured to powderize the purified and sterilized silkfibroin-based solutions to obtain the silk fibroin in a powder form.Many modifications and other implementations of the disclosure will cometo mind to one skilled in the art to which this disclosure pertainshaving the benefit of the teachings presented in the foregoingdescriptions and the associated figures. Therefore, it is to beunderstood that the disclosure is not to be limited to the specificimplementations disclosed herein and that modifications and otherimplementations are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

FIGS. 9A-9C depict different configurations (top and front views) ofscreens 930, 930′, 930″ that may be integrated into the systemsdisclosed herein, for example, within the first processing substationgenerally or the reactor vessel specifically, to, for example, helpcontrol movement of the cocoons. The screen 930 depicted in FIG. 9Aincludes a ring 988 with a conical shaped basket 990 extendingtherefrom. The basket 990 includes a plurality of perforations 992 thatpermit flow therethrough (e.g., liquids, possibly along with othercomponents that are smaller than the perforations and typically deemeddesirable and/or insignificant). Generally, the ring 988 may beconfigured to provide an interface to the equipment (e.g., attachment tothe bottom of the vessel via a flange assembly), support to the basket990, and/or means for handling the screen 930. The screen 930 of FIG. 9Aincludes an optional handle 994. The screen 930′ depicted in FIG. 9Balso includes a ring 988′ and a basket 990′ extending therefrom, wherethe basket 990′ has a generally tapered or frusto-conical shape (alsoknown as a Pilgrim’s Hat) and also includes a plurality of perforations992′ formed therein. The screen 930″ depicted in FIG. 9C also includes aring 988″ and a basket 990″ extending therefrom, where the basket 990″has a generally cylindrical or oblong shape and includes a plurality ofopenings 992″ formed therein. In some embodiments, the basket 990″ maybe constructed from a woven mesh screen, with the wire size, spacing, %open area, etc. selected to suit a particular application as disclosedbelow.

Generally, the screens 930, 930′, 930″ can be used to ensure thatundesirable aspects of the silk fibroin solution do not flow through theoutlet (928 a in FIG. 10 and 1028 a in FIG. 11 ), including silk fibroin(including fragments) and debris included within the silkworm cocoons(e.g., silkworms, organic material (plant material, soil, etc.),inorganic material (packaging, ties, etc.)). It should be understoodthat the sizes, shapes (cylindrical, rectangular, bowl, etc.),perforation type, opening sizes, open area, and distribution (e.g.,whether perforations 992, 992′, 992″ are disposed on the entire surfaceof the basket), and materials (e.g., stainless steel, polymers, etc.) ofthe screens can be changed depending on the desired location and use ofthe screens, and whether the screens 930, 930′, 930″ will be cleanedduring processing. For example, a screen with larger perforations may beused in tandem with a screen with smaller perforations to removedifferent materials at different stages of the process and/or differentlocations along the flow path, as shown in FIGS. 10 and 11 .

FIG. 10 depicts one embodiment of a first processing substation 920(with or without agitation equipment 934), where a single screen 930 ispositioned at the bottom of the reactor vessel 922 just before theoutlet 928 a. FIG. 11 depicts another embodiment of a first processingsubstation 1020 (with or without agitation equipment 1034), where twoscreens 1030′, 1030″ are utilized. Specifically, a first screen 1030″ ispositioned at the bottom of the reactor vessel 1022 just before theoutlet 1028 a, while a second screen 1030″ is positioned downstream ofthe outlet 1028 a, for example, secured within the piping exiting thevessel 1020 via a pair of tri-clamps 1096 or similar mechanism thatpermits for easy removal of the screen 930″ for cleaning or replacement.Generally, the screens 930, 930′, 930″ may be secured via flanges(preferably with gaskets), threaded connections, or other suitablemeans. Additionally, one or more valves may be incorporated to isolate ascreen and and/or provide for easy removal from the system. Aspreviously mentioned, the screens 930′, 930″ may have differentperforation schemes, shapes, etc. to suit a particular application. Forexample, the screen 1030′ disposed within the vessel 1022 may comprise acourse mesh that clogs less frequently, requiring fewer cleanings andproviding faster draining, while the screen 1030″ disposed downstream ofthe vessel may comprise a finer mesh, but is easily removed for cleaningor replacement. Generally, placement of the screens may be selected tosuit a particular application (e.g., ease of insertion and removal, fitwithin the vessel, mounting configuration, etc.).

What is claimed is:
 1. A silk manufacturing system comprising: a first processing substation comprising at least one reactor vessel configured to receive silk inputs, extract silk fibroin proteins therefrom, and produce a silk fibroin-based solution, such that the silk fibroin-based solution is substantially free of sericin, wherein the first processing substation is configured to extract the silk fibroin proteins via degumming, rinsing, and dissolving processes within a single one of the at least one reactor vessel; and a second processing substation in fluid communication with the first processing substation, the second processing substation configured to receive and purify the silk fibroin-based solution from the first processing substation, wherein the second processing substation comprises a filtration module comprising at least one membrane configured to substantially remove the second compound and produce a purified silk fibroin-based solution.
 2. The system of claim 1 further comprising a heat exchange system configured to adjust a temperature of the silk fibroin-based solution prior to or after any one of the processing substations.
 3. The system of claim 1, wherein the second processing substation is configured to purify the silk fibroin-based solution via diafiltration.
 4. The system of claim 1, wherein the second processing substation is configured to purify the silk fibroin-based solution via tangential flow filtration.
 5. The system of claim 1, wherein the purified silk fibroin-based solution comprises less than about 650 ppm of the one or more salts or non-organic particulates.
 6. The system of claim 1 further comprising a third processing substation in fluid communication with at least one of the first processing substation or the second processing substation the third processing substation configured to receive and powderize the silk fibroin-based solution.
 7. The system of claim 1 further comprising a fourth processing substation in fluid communication with the second processing substation, the fourth processing substation configured to receive and sterilize the purified silk fibroin-based solution.
 8. The system of claim 7, wherein the fourth processing substation comprises a microfiltration module.
 9. The system of claim 8, wherein the microfiltration module comprises: a first microfiltration stage having a pore size between about 0.7 µm and about 5 µm; and a second microfiltration stage disposed downstream of the first microfiltration module, the second microfiltration stage having a pore size between about 0.05 µm and about 0.8 µm.
 10. The system of claim 8, wherein the microfiltration module further comprises one or more holding tanks, wherein the tanks may be configured to provide additional processing, including one or more of storing the solution, temperature control of the solution, or adjusting the solution concentration to address turbidity or sterility levels.
 11. The system of claim 6, wherein the third processing substation comprises a spray dryer.
 12. The system of claim 6, wherein the powderized silk fibroin-based solution has a water activity level of less than 0.9.
 13. The system of claim 6 further comprising a post-treatment system configured to receive a silk fibroin powder from the third processing substation and to at least one of: condition the silk fibroin powder, test the silk fibroin powder, or package the silk fibroin powder in a food-safe container.
 14. The system of claim 1, wherein the at least one reactor vessel configured to receive silk inputs is further configured to operate with a packing density thereof of about 1% and about 30%.
 15. The system of claim 1, wherein the at least one reactor vessel is sized to have an aspect ratio of height to diameter as defined by a work volume of about 0.5 to about 5.0.
 16. The system of claim 1, wherein the at least one reactor vessel further comprises a handling structure for controlling at least one of movement or position of the silk inputs within the at least one reactor vessel.
 17. The system of claim 1 further comprising a pre-treatment system configured to condition the silk inputs prior to or at introduction to the first processing substation.
 18. The system of claim 1, wherein the silk inputs come from a Bombyx mori silkworm.
 19. The system of claim 1 further comprising: a reservoir disposed between the first and second processing substations and configured to at least one of hold or condition the silk fibroin-based solution; and a pump assembly disposed between the first and second processing substations and configured to transfer the silk fibroin-based solution between the first processing substation, the reservoir, and the second processing substation.
 20. The system of claim 1, wherein the at least one membrane is a spiral wound membrane.
 21. A silk manufacturing system comprising: a first processing substation comprising at least two single reactor vessels configured in parallel, each vessel configured to receive silk inputs, extract silk fibroin proteins therefrom, and produce a silk fibroin-based solution, such that the silk fibroin-based solution is substantially free of sericin, wherein the first processing substation is configured to extract the silk fibroin proteins via degumming, rinsing, and dissolving processes within each vessel; and a second processing substation in fluid communication with the first processing substation, the second processing substation configured to receive and purify the silk fibroin-based solution from the first processing substation, wherein the second processing substation comprises a filtration module comprising at least one membrane configured to substantially remove the second compound and produce a purified silk fibroin-based solution.
 22. A silk manufacturing system comprising: a first processing substation comprising at least one reactor vessel configured to receive silk inputs, extract silk fibroin proteins therefrom, and produce a silk fibroin-based solution, such that the silk fibroin-based solution is substantially free of sericin, wherein the first processing substation is configured to extract the silk fibroin proteins via degumming, rinsing, and dissolving processes within a single one of the at least one reactor vessel.
 23. The system of claim 21 further comprising a third processing substation in fluid communication with at least one of the first processing substation or the second processing substation, the third processing substation configured to receive and powderize the silk fibroin-based solution.
 24. The system of claim 23, wherein the third processing substation comprises a spray dryer.
 25. The system of claim 23, wherein the powderized silk fibroin-based solution has a water activity level of less than 0.9.
 26. The system of claim 21, wherein the second processing substation is configured to purify the silk fibroin-based solution via diafiltration.
 27. The system of claim 21, wherein the second processing substation is configured to purify the silk fibroin-based solution via tangential flow filtration.
 28. The system of claim 21, wherein the at least one membrane is a spiral wound membrane. 